Two years after the events in The Bourne Identity, Jason Bourne (Matt Damon) and his girlfriend, Marie Kreutz (Franka Potente), are living on the beach in Goa, India. Bourne is beginning to recover some of his memories, and he is troubled by disjointed flashbacks of an assassination he carried out in a Berlin hotel.
Meanwhile, in Berlin, a CIA officer working for Deputy Director Pamela Landy (Joan Allen) is arranging to pay $3 million in exchange for the "Neski Files", documents pertaining to the theft of $20 million from the CIA, seven years earlier.
During the exchange, a Russian assassin named Kirill (Karl Urban) intercedes. He plants two explosives in the basement electrical circuit, one on the main and the other on a sub-line. He tags one of the items with a fingerprint belonging to Jason Bourne. The explosive on the main line kills the power, while Kirill kills both the agent and the person with the files, then steals both the files and money. He gives those to Russian oil magnate Yuri Gretov (Karel Roden).
Kirill then travels to Goa, his next task being to kill Bourne, but Bourne sees Kirill first and recognizes he's out of place. He gets Marie and tells her they have to get out fast. As they take off, Kirill sees them, gets in his car, and gives chase. Bourne gets Marie to switch places with him and take over the driving. Not long after that, he prepares to get out of the car, instructing Marie to drive back to their cottage on the beach and he'd meet her there in one hour.
Kirill gets hung up in traffic and bails out of his car, grabbing his rifle from the trunk. As Bourne and Marie are crossing a bridge, in heavy traffic, Kirill fires his sniper rifle and hits Marie. She loses control of the car and it careens through a guardrail and off the bridge into a river. Bourne gets out of his side of the car and swims around to get Marie out. He takes a gulp of breath from air trapped near the roof of the vehicle, then attempts mouth-to-mouth on Marie there, underwater. He realizes she is dead, and lets her go. She slowly drifts away in the current.
Kirill looks down at the water from on the bridge, then leaves, believing that he had killed Bourne. He later meets up with Gretov, confirming that Bourne was dead, and collects his payment.
Bourne managed to swim away from the scene, undetected. He returns to his cottage and collects and burns all of Marie's passports and pictures, except for one picture showing them in an embrace. He collects all of his passports, cash, and the notes that he used to dictate his dreams, then leaves India. Bourne travels to Italy to learn why it is he is being targeted again.
Landy's team investigates the scene of the killings in Berlin and finds the fingerprint that Kirill planted. Checking various fingerprint databases, they determine that it is linked to someone who worked at the CIA, specifically with the secret Treadstone project, which was top secret, requiring a level 5 clearance to read about.
Landy flies to CIA headquarters in Langley, Virginia to find out more information. She meets with her superior and asks for the level 5 clearance to continue her investigation. He approves it.
Landy learns that the fingerprint belongs to Jason Bourne. She notes the other names of personnel listed in the files and decides to contact CIA Chief Ward Abbott (Brian Cox), asking to meet with him. When they meet, Abbott denies knowing anything about a program called Treadstone. He quickly learns she Landy knows all about it, having acquired the level 5 clearance, including that he was very much involved, so he tells her that as far as he knows, Jason Bourne was long dead. When she tells him about the men killed in Berlin recently, and Bourne's fingerprint found at the scene, he appears to become very concerned.
Based on what was found on the hard drive of a laptop computer belonging to the deceased Alexander Conklin, the former head of Treadstone, Landy tells the people she's working with, including her boss and Abbott, that Bourne and Conklin may have been involved in some sort of lucrative venture together (Conklin had a personal bank account with $760,000 in it), and that Bourne appeared to still be involved in whatever it was, so that's why he'd resurfaced. While Landy is briefing her superior and the other staff, an agent comes in and says Bourne's passport showed up in the system in Naples, Italy.
Landy tells Abbott that the stolen files contained information about the CIA agent who was suspected to have stolen the $20 million from the CIA, and there was a Russian politician, Vladimir Neski, who was prepared to identify the thief, but before he could do so, he was killed by his wife in a Berlin hotel, in a murder-suicide. Based on the fingerprint, Landy believes Bourne killed the CIA officer and the source in Berlin, so Abbott and Landy set out to capture him.
When Bourne arrives in Naples, Italy, he is detained by an Italian Carabinieri officer and CIA interrogator, John Nevins (Tim Griffin). Bourne stays mute, frustrating Nevins. Tom Cronin (Tom Gallop) calls from the CIA in Langley and tells Nevins that Bourne is an agency priority target. Bourne senses trouble from what he hears Nevins say, and responds intuitively, reacting before Nevins can bring his gun to bear, taking down both Nevins and the officer in the room.
After quickly copying the Nevin's phone SIM card onto his own cell phone, Bourne escapes. That SIM allowed Bourne to listen in on a call to Nevin's from Landy and he learns her name and phone number, then finds out that he is suspected in the recent killings in Berlin.
As Landy is planning to get on a plane to Europe, Abbott warns her that she's getting into more than she can deal with. Cronin, their boss, orders Abbott to go with her and to put a stop to Bourne's activities.
Bourne steals a car and heads to Munich. As he drives, he has more flashbacks, involving Conklin and a man and woman he'd shot to death.
In Amsterdam, Netherlands, Abbott finds Nicky Parsons (Julia Stiles), the gal who worked in Paris for three years. She was specifically tasked with monitoring the agents assigned to Treadstone. Landy forces Nicky to go to Italy with Abbott and Landy, considering her critical to their attempts to capture Bourne.
In Berlin, the CIA office has video of Bourne at the airport. One agent figures Bourne has made a big mistake by being so careless, but Nicky tells him that Treadstone agents don't make mistakes, that if Bourne is able to be tracked, it's because he wants to be.
Bourne finds and interrogates the only other remaining Treadstone agent, Jarda (Marton Csokas), who tells him that the project was shut down following Conklin's death. Jarda most likely had been forewarned about Bourne, and they end up having a huge fight, in which Bourne emerges victorious, by strangling Jarda to death. Bourne then creates a leak in the house's natural gas supply and puts a magazine into the toaster to slowly heat up and create a flame. The subsequent explosion slows down arriving police and allows Jason to escape.
Bourne searches hotels in Berlin until he finds where Pamela Landy is registered. He watches until she leaves and follows in a taxi. He goes to the top of a building, opposite of the one she enters, and sets up a rifle and scope. With Landy in his sights, he dials her cell phone and asks her if she's running Treadstone. She tells him Treadstone had been shut down, and he knew it. She then asks him if he remembers killing two people in Berlin.
Bourne tells Landy he wants to come in. He then sees Nicky come into view and tells Landy that he wants them to send "that girl from Paris" to meet him and bring him in. When Landy asks what if she can't find that girl, Bourne says, "she's standing right next to you." That freaks them out as they immediately look out the windows to try and pick up on where he is.
Abbott and Nicky want snipers positioned to kill Bourne when he meets with Nicky. Landy wants him alive, so she can learn what he knows about the Neski case, but after Abbott has a private conversation with her, she does decide to go ahead and position the snipers.
Nicky is given Landy's cell phone, per Bourne's instructions. Bourne calls and directs Nicky to board a streetcar, which he's also on. He escorts her off the streetcar before any of the tailing CIA men can get to her, and before any of the snipers can see him. He takes her down into the subway, to a room out of public view, interrogating her along the way, and inside the room.
Nicky is able to prompt a bit more memory in Bourne's mind, about a Russian politician named Neski. When he asks her when he was in Berlin, she tells him he was never in Berlin. He is convinced she's lying and becomes so upset that he nearly shoots her, but he also senses that she's telling the truth, as far as she knows things to be, so he ends up leaving her and taking off again.
Upon overhearing the conversation between Nicky and Bourne (Nicky was wired), Landy begins to believe that Bourne really can't remember killing the two men in Berlin, or that maybe he didn't actually do it. Abbott becomes increasingly agitated that Bourne can't be caught or killed.
Bourne goes to a library and finds articles about the Neski murders, which occurred at the Brecker Hotel in Berlin. He decides to go there.
Abbott's young associate, Danny Zorn, takes Abbott down to the electrical breaker room, in the Berlin building where the killings occurred, and shows him how the explosive charges that were set were done in a way that was unnecessary, and that he thinks Bourne wouldn't have done such a thing, therefore, it's possible that Bourne and Conklin were being framed. Abbott asks Zorn to show him how the explosives were set again. When Zorn was focused on the electrical connections again, Abbott grabbed him and choked him to death.
Bourne arrives at the Brecker Hotel and goes to the room of the murder. He is able to remember being sent to kill Neski, on Conklin's orders, and that Neski's wife unexpectedly showed up. He remembers shooting them both, then putting the gun in the wife's hand to make it look like a murder-suicide.
Bourne is able to escape the police, who'd been called after a desk employee recognized him from a wanted poster, by climbing up a firewater pipe outside the building. The police on the street see and recognize him and give chase. He barely evades capture several times, eventually jumping off a bridge onto a river barge, hurting his left leg, then uses a grappling hook to pull himself up onto another bridge. He is able to limp over to a streetcar and make his escape.
Abbott is shocked to learn that Bourne let Nicky go, unharmed. He feigns concern to an associate about where Zorn might be, and wonders aloud about why Bourne might have come to the Hotel Brecker. Landy recognizes the room Bourne was in as being the Neski murder room. She no longer believes that Bourne killed the Neskis. She receives word that Danny Zorn's body was found.
Abbott calls Gretkov and tells him that his CIA associates are on to the facts about Neski. He emphasizes that Gretkov must kill Bourne, or their involvement in the theft of the $20 million would be revealed. Gretkov tells Abbott that he isn't obligated to do anything anymore, and hangs up on Abbott. Bourne then appears behind Abbott.
Abbott admits everything to Bourne. Bourne thrusts his pistol up against Abbott's head and demands to know why they just couldn't leave him alone after Treadstone ended. Abbott tells Bourne that it's just the way things had to be, that Bourne was a killer and always would be, so he likely wouldn't ever be left completely alone.
Abbott tells Bourne to go ahead and shoot him, but Bourne says Marie wouldn't want him to, so it's because of her that he didn't shoot. He then shows Abbott that he has been recording the conversation. Before Bourne leaves the room, he sets a pistol on the desk next to Abbott.
Landy walks in on Abbott to find him holding the pistol, pointed at her. Abbott tells her that the work he did for the CIA was a result of his patriotism. When she asked about Danny, Abbott called that "collateral damage." When she asks Abbott what they do now, he turns the pistol on himself and shoots.
As Landy listens to the tape recording that Bourne got to her, she continues to work with her people to try and track him down. He's headed for Moscow, where Gretkov is in the process of tracking down Kirill to let him know Jason Bourne was still alive.
Bourne takes a taxi to the address of Vladimir Neski's daughter. A woman was outside sweeping the front steps, and Bourne approaches her. She tells him that the Neski girl had moved, and she gives him the new address. Meanwhile, the police are aware that Bourne's in Moscow and are busy passing around wanted posters. One of the other taxi drivers recognizes him and tells police which taxi driver he went with.
The police, and Kirill, simultaneously close in on the neighborhood where Bourne is. Bourne hears the sirens and begins walking. Kirill sees him first, jumps out, and shoots, hitting Jason on top of his shoulder. The police arrive and detain Kirill, not knowing who he was, only that he was shooting at somebody. That gives Bourne time to slip away again.
When Kirill shows his i.d. to the police, he is let go and he again takes off after Bourne, this time on foot. Some other policemen on the street recognize Bourne as he enters a market to get some items for his wound. Bourne forces one of the cops down to the floor and walks out of the market. As some other police approach him outside, he knocks them out and steals a taxi cab. Kirill sees what's happened and commandeers a vehicle from woman and gives chase.
As Jason drives away, he checks a map he stole at the market to get a quick idea of the road patterns in the area, and where it was he needed to go. He then has to focus his full attention to his pursuers, which are many, in the subsequent furious and violent car chase.
The chase reaches it's climax when Bourne and Kirill are slamming their vehicles into each other, side by side, as they travel at high speed in a congested tunnel. Bourne shoots out the tires of Kirill's vehicle, causing it to turn sideways. Bourne's vehicle t-bones it and pushes it into a concrete support divider. He gets out of his car with his pistol at the ready, but doesn't need to shoot, as Kirill is extensively injured and dying.
Gretkov is arrested by Russian police, after Landy provides them with the evidence she got from Bourne.
Bourne continues on to find Irena Neski (Oksana Akinshina). He breaks into her apartment and is waiting when she comes home. He is holding his gun, has blood on his hand, and is sitting in a chair in the dark. When she turns on the light and sees him, she is obviously frightened, but he tells her to not scream, and that he won't hurt her.
Bourne tells Irena that she's older than he thought she'd be. He knows she has been living with the belief that her mother killed her father and then killed herself. Bourne has tears in his eyes as he tells Irena that it was him who killed her parents. He tells her it was his job, his first time, and that her father was supposed to be alone. When her mother appeared, he had to change his plan. He tells her he's sorry. Irena has tears in her eyes, but doesn't speak as Bourne quietly walks out of her apartment.
In New York City, Bourne contacts Landy to ask why the CIA was still looking for him. Landy thanks Bourne for supplying the tape, which the CIA had used to settle matters concerning his frame-up. She apologizes to Bourne (off the record), then tells Bourne that his real name is David Webb, and that he was born in Missouri on April 15, 1971.
She wants him to come in so they can talk more about that. Bourne declines Landy's offer, then tells her she should get some rest, as she looks tired. She turns to look out the window, but he's gone, out into the crowded streets.