The film begins with a turtles prologue, explaning their transformation and their victory of defeating their arch-nemisis The Shredder. Then, 3000 years back, Yaolt, a warrior-king, and his four generals, lead an army in conquest of the world. After defeating a Central American kingdom, Yaotl learns that when nine stars align every 3000 years, called the Stars of Kikan, a portal of immense power to another world opens as a beam of light. The portal gives Yaotl immortality, but turns his generals to stone and releases thirteen monsters into the world, which decimates his army and remain active into the present.
In present day Central America, a group of corrupt army officers steal a village's supplies. While driving through a forest, the group is stopped by a log on the road and is silently attacked by an unseen enemy. Their truck and the village's supplies roll back to the village the next morning. Some time later, April O' Neil searches through the forest to discover Leonardo.
Leo asks of his brothers, and what has become of them after his departure. Donetello is now a one-turtle tech-support company, Michealangelo has started a party buisness called Cowabunga Carl, and Rapheal sleeps all day, and by night is a motorcycle riding vigilante named The Nightwatcher, where he is taking out robbers who have robbed a bank.
April, meanwhile, has started a shipping business and was in the country to get an ancient artifact. Leo has been living in Central America for the past year in order to find himself as a leader, and he doesn't want to return lest Splinter think him a failure. April pleads with him to come back, but fails. Though she suspects this since he disappears before she can say anything else.
Back in New York, Mikey drives his Cowabunga Carl van through the streets and parks it in an alleyway. Slipping through a manhole, he skates the sewer pipes to the turtles' lair. While watching a story about Nightwatcher, Mikey shows his fondness for his vigilante days, which Don tells him to get over. Raph mocks him for moving on so quick and supports the Nightwatcher, which starts an argument between the two. Master Splinter comes in and stops the argument from escalating, and Ralph heads off to bed.
At the docks, April is supervising the unloading of an ancient statue for her cargo business. After calling her roommate Casey Jones, the two bring the statue to their client, Max Winters. Winters is the head of WintersCorp, one of the largest corporations on Earth. Casey accidentally knocks over a ceramic pot, which triggers an alarm that locks down the whole building. The statue is the last of the Four Generals, which Winters has been gathering. After April and Casey leave, Karai (Zhang Ziyi) and the Foot Clan enter. After the defeat of the Shredder, the Foot Clan has lost a great deal of its former glory, agreeing to work for Winters for a price. Winters orders them to be his eyes and ears and to bring to him some "friends" who will be coming to the city.
As Nightwatcher, Raphael is confronted by Casey, who knows it's Raph under the armor, saying that he looks like a big, metal turtle. The two join forces to catch a crook that Raph busted a week ago. Later that night it is made clear Raph does miss Leo, despite his anger towards him, while Casey admits his own insecurities over his and April's relationship. In Winter's tower, Winters uses small devices to wake his Stone Generals up.
Elsewhere, Leo makes his way into New York through holding an airplane tire. He leaps off and lands in the water, and soon is making his way through the sewers. He enters the Turtles' base and finds an unsurprised Splinter in his dojo. Leo apologizes for failing, and Splinter assures that he didn't. It's more important to focus on making his family a team again and he forbids the four from fighting until they can fight as one. Splinter also mentions to Leo that Raph was the most hurt by his absence, although he won't admit it. Raph hears this and becomes jealous of Splinter's favor of Leo over him. Leo wakes up Don and Mikey, and they are overjoyed to see him, while Raph briefly greets him before going to bed.
Later, as the four turtles meet on a building to train, where Leo admits he wants to stop the Nightwatcher, the four discover the Foot Clan fighting a giant, ape-like monster in a building construction. In order to stop it from escaping, the four join in and cause extreme damage to the building before being covered in rubble. While the brothers are buried, the four stone generals appear, defeat the monster, and carry it off in a garbage truck. The turtles free themselves and return to the sewers.
In the WintersCorp building, Karai is angry that she was not told of the monsters, but agrees to continue assisting the stone generals, even after Winters apologizes for making them 'hired guns', and stating that he has more faith in his Stone Generals. The monster is placed in chains and sealed in a cage. After learning of the incident, Splinter reprimands the turtles for fighting against his orders. Raph becomes angry at Leo and leaves the lair. The Stone Generals, meanwhile, along with the Foot, violently defeat the monsters one at a time.
Raph meets up with Casey and the two witness the Foot and the Generals beat a bat-like monster. They are spotted and Raph is hit with a dart and a General's stone shuriken before they escape. One of the Stone Generals, Gato, goes after them, but runs off to avoid being seen by a police helicopter. Raph falls unconscious afterwards.
Meanwhile, the generals are beginning to realize that once Winters has all thirteen monsters, their immortality will be forfeited, so they begin laying plans against him while Don calculates that once the stars align, the portal will fall right into the WintersCorp building.
That night, Raph, as Nightwatcher, saves a cook from another monster and is attacked by it in the diner, soon feeding it the ninja weapon smoke bomb. The monster runs out, and the Nightwather is mistaken by the cook as a villain, as he tries to give him back the cash register, but is seen by Leo who chases him down. Eventually they fight on a rooftop and Raph's helmet is knocked off by an Upper cut from Leo. The two argue before Raph challenges Leo to prove his superiority. In the fight, Raph breaks Leo's swords and wins, but, realizing how close he came to killing his brother, runs off. As Leo watches him go he is hit with a dart by the Generals, who wish to use him as a substitute for the final monster. Raph hears his screams but is too late to help.
Raph heads back home and confesses what he's done to Splinter, telling his master that he understands why he chose Leo to lead the four of them. Splinter assures him that he respects Raph's passion and courage, the traits of a great leader, and he does love him deeply, also stating that these traits must be tempered with compassion and humility. Raph tells Splinter that Leonardo has been captured, showing him the broken swords that he himself broke. Soon the whole team, including Casey and April, led by Raph, make their plan to get Leo back. April reveals a new yellow ninja outfit she got in Japan, and gives Casey a black, metal hockey mask.
The stars align and a beam of energy shoots from the sky and down the roofless tower of Winter's building. Inside, Winters is starting up the process when things start going wrong and as the battle between an army of Foot and the Turtles rages outside, the generals tell Winters that they have no plans on becoming mortal again.
The Turtles get into the building and shut the security doors to keep the Foot out. This is accomplished by Casey, who breaks another ancient looking piece of pottery, closing the heavy metal doors. They free Leo and Raph gives him a new pair of swords, tucked in Casey's weapons bag, as an apology. Winters gets tossed off the balcony in his corporations main hall, by his betraying stone brothers, and wakes up to see April and 'giant turtles' standing before him. At first he thinks he's simply dazed, until April tells him they know what he's been up to, and wants him to close the portal. Winters tells them that is exactly what he is trying to do, to send the monsters back and break the curse he and his brothers are under, right as the Foot break the door down.
The Generals then reveal there plans to use the portal to summon an army of monsters forth into their world, and keep their immortal curse going as long as they don't have the 13th monster. They urge the Foot to join them, but Karai honors her agreement with Winters and sends her ninjas to locate the final monster so that they can bring it back to WintersCorps tower. The turtles stay behind to deal with the generals, Splinter and Winters fight the monsters that are coming out of the form the portal back, and April, Casey, and Karai go to find the real thirteenth monster.
Working as a team, the Turtles prove to be an even match for the Generals. Leonado battles the cat-like General Gato, Michelangelo battles the female snake-like General Serpiente, Donatello battles the gorilla-like General Mono, and Raphael battles the eagle-like General Agulia. Impressed with their skills, Agulia offers them a place along side them, which Raph naturally rejects.
Pretty soon, Michelangelo's 'Cowabunga Carl Party Van', driven by April, who is arguing with Casey, is seen rocketing through the streets with a dragon on its tail, while Karai wonders why they aren't more concerned about the monster chasing them. April drives the van through the lobby just as the turtles get all four generals lined up. April brakes hard, crashing into various objects and causing the chasing dragon to trip and slide across the marble floor, sweeping the generals into the vortex, sending both the generals and the monsters to another world.
As the team pulls itself clear of the rubble, Casey kisses April, much to Raph's amusement. Karai tells the turtles that they will have business later and hints at the return of the Shredder before leaving. Winters, laughing joyously, dies, turning into dust which Mikey accidentally breathes in.
Back at the headquarters, Splinter puts Winter's battle helmet on a shelf in his dojo, along with the Shredder's helmet. Raph also puts his Nightwatcher helmet on the shelves, retiring it from service. Mikey soon attempts to put his "Cowabunga Carl" mask on the shelf. That night, the turtles jump across the rooftops of New York as Raph reflects on what it means to be a hero... and what it means to be family.