Under the watchful eyes of Father Silvano Bentivoglio and Dr. Vittoria Vetra, the European Organization for Nuclear Research (CERN) initiates the Large Hadron Collider and captures three vials of antimatter. Immediately afterward, someone kills Father Silvano, using his retina to infiltrate the containment chamber, and steals one vial.
The Roman Catholic Church mourns the death of Pope Pius XVI in Rome. Vatican City prepares for the College of Cardinals' papal conclave, which will select the next Pope. Until that time, Camerlengo Patrick McKenna, a papal court official and former helicopter pilot, assumes temporary control of the Vatican. Reporters, nuns, priests, and other faithful members of the Church crowd into Saint Peter's Square, waiting for the white smoke from the conclave, signalling a successful vote. But the Illuminati, a 400-year old, underground secret society, kidnap the four most likely candidates before the conclave enters seclusion. The Illuminati threaten to kill one every hour, beginning at 8:00 pm, and then destroy the Vatican in a burst of light at midnight. A stolen security camera shows the missing antimatter vial, which will catastrophically explode when the vial's battery dies and the magnetic containment field fails.
The Vatican summons symbologist Robert Langdon from Harvard University and Vittoria Vetra from CERN to help them solve the Illuminati's threat, save the four preferiti, and replace the vial's batteries. Langdon listens to the Illuminati message and deduces that the four cardinals will die at the four altars of the "Path of Illumination." However, no one knows where these altars are located. Vetra demands that Commander Richter, the commandant of the Swiss Guard, to bring Father Silvano's diaries from Switzerland, hoping that they contain the name of the person with whom Silvano discussed the antimatter experiment. Langdon also demands access to the Vatican Secret Archives (something he has requested for 10 years) to see the original copy of Galileo Galilei's banned book, which may contain the locations of the four "altars of science." Using the clues from this book, Langdon, Vetra, Inspector General Ernesto Olivetti, and Lieutenant Valenti of the Vatican Gendarmerie Corps race to the first church, only to find the first cardinal, Cardinal Ebner, dead, suffocated with dirt, eaten by rats and branded with the word "Earth." They verify the second altar's location and arrive, only to witness the death of the second cardinal, Cardinal Lamassa, his lungs lacerated and his body branded with the word "Air." While Vetra studies Silvano's diaries, Langdon and the Vatican officers locate the third church and try to save the third cardinal, Cardinal Gudiera, from burning to death, but the assassin appears and kills everyone but Langdon. The cardinal succumbs to the flames, his body branded with the word "Fire."
After escaping, Langdon convinces two Carabinieri officers to race with him to the last church of the "Water" altar, but the assassin murders the officers and drops the fourth cardinal, Cardinal Baggia, into the Fountain of the Four Rivers. However, Langdon saves the cardinal, who tells him the location of the Illuminati's lair: Castel Sant'Angelo. When Langdon and Vetra arrive, they are confronted by the assassin, who spares their lives since they are not armed and he has not been paid to kill them. He reveals that his contractors were from the Catholic Church. The assassin escapes and finds a vehicle containing his payment, but is killed by a car bomb upon igniting the engine. Langdon and Vetra discover that the final victim of the plot will be Camerlengo McKenna. After arriving at the Vatican via a secret passage, they and some Swiss Guards enter the Camerlengo's office and find him in the floor branded with the Vatican's symbol on his chest and Commander Richter near him with a gun. The Guards promptly kill Richter to save the camerlengo. During the confusion, the dying commander gives Langdon a key to his office. Then the camerlengo, Langdon, Vetra, and the Swiss Guards discover the location of the stolen antimatter vial. By the time they find it, the battery is about to expire, the deadly explosion just minutes away. The camerlengo seizes the vial and uses a helicopter meant for escape from the Vatican to fly above the church. He then activates the autopilot and escapes with a parachute. After several seconds, the bomb explodes and the camerlengo lands, now considered a hero by the crowd and even as the best candidate to be the new Pope by the College of Cardinals. Meanwhile, Langdon and Vetra use Richter's key to watch a security video showing that the mastermind behind the murders of the original Pope and the preferiti and the antimatter robbery, in fact, is the camerlengo and not the Illuminati. While Richter tries to arrest McKenna, the priest brands himself with a seal that resembles Saint Peter's upside-down crucifixion and accuses the commander being a member of the Illuminati. Langdon shows the video to the College. After the camerlengo realizes his plot has been uncovered, he immolates himself with oil from one of the 99 holy lamps inside St. Peter's Basilica.
The Vatican announces that the camerlengo died due to internal wounds suffered during his landing, while the public demands he be canonized. The College designate the Cardinal Baggia as the new Pope (who chooses to take on the name Luke), and Cardinal Strauss as the new camerlengo. The new camerlengo thanks Robert Langdon for saving the Vatican and the new Pope, and as a mark of his gratitude loans Galileo's "Diagramma Veritas" to Langdon for his reference. The film ends with the new Pope walking out on the balcony to the cheering crowd in St. Peter's Square.