The movie begins immediately following the car chase near the end of The Bourne Supremacy, with a wounded Jason Bourne evading the Moscow police. Cornered by police while breaking into a medical clinic to treat his wounds, Bourne shows his human side by allowing a policeman to live during a tense confrontation, before making his getaway.
The story continues six weeks later when Bourne goes to Paris to tell Marie's brother of her death. On the way, Bourne reads an article in The Guardian where journalist Simon Ross describes Jason Bourne as a CIA operative. Bourne arranges to meet Ross in London at Waterloo station. However, Ross is now under CIA surveillance, after his mention of Blackbriar in a phone call to his editor alerted the CIA via Echelon. The CIA follow Ross to Waterloo Station, believing him to be meeting his source there. At the station, Bourne sees the CIA agents following Ross. Bourne plants a prepaid mobile phone on Ross and calls him, instructing Ross on how to evade the agents and knocking out any others tailing them. However, CIA official Noah Vosen, who heads Operation Blackbriar (a renamed Treadstone proposed by Ward Abbott at the end of The Bourne Identity), orders an assassin named Paz to kill Ross and his source. Vosen's team sights Bourne on a security camera, and recognize him as a Treadstone assassin, and assume that he must be Ross's source. Bourne keeps advising Ross on hiding, but a panicked Ross reveals himself, giving Paz a clear killing shot. In the ensuing chaos, Bourne steals Ross's notes from his body. The notes reveal Ross's source as Neal Daniels, the CIA's Madrid station chief.
Deputy Director Pamela Landy is brought in to help capture Bourne. With her help, Vosen and his team realize that Daniels was the source. They send a team to his office, but Bourne arrives first, defeating the CIA team when they come. While there, Nicky Parsons turns up. Nicky tells him that Daniels fled to Tangier and helps him escape the CIA reinforcements. When Bourne asks Nicky why she is helping him, Nicky only hints vaguely to something Bourne has forgotten due to his amnesia. In Tangier, they realize that the CIA has sent assassin Desh Bouksani for Daniels. Nicky uses her official clearance to send Desh a message, telling him to meet her for a new phone. This allows Bourne to follow Desh to his target. When Vosen realizes Nicky's deceit he orders Desh to kill Nicky and Bourne after Daniels. Landy is outraged at Vosen's willingness to indiscriminately kill CIA personnel and thus quits the operation.
Bourne follows Desh, but fails to prevent Daniels' death. When Desh returns for Nicky, Bourne outruns the Tangiers police and fights Desh, eventually strangling him with a towel. At the Tangier morgue, Bourne examines Daniels' charred papers and finds the address of the CIA substation in New York City. While Bourne journeys to the U.S., Nicky has no choice but to go into hiding. In New York City, Bourne calls Landy while observing her and Vosen from across the street. The ending of The Bourne Supremacy is repeated: Landy tells him his real name is David Webb and that he was born on "4/15/71", a code for the address of the Treadstone facility at 415 East 71st Street. Bourne sends a text message to Landy to arrange a meeting, and Vosen and his team, who have intercepted the message, follow as she leaves the building. This is actually a diversion, however, allowing Bourne to enter Vosen's office and steal classified Blackbriar documents.
Bourne's journey to the facility is delayed by a car chase with Paz, whom Bourne chooses not to kill, given the chance. Just outside the facility, Bourne meets Landy and gives her the stolen documents before entering the building. Inside, he meets Dr. Albert Hirsch, who ran Treadstone's psychological conditioning. With his help, Bourne remembers that as Captain David Webb, he volunteered for the program. However, he tells Hirsch that he no longer wants to be Bourne or have anything to do with Treadstone and the CIA. Back at the substation, Vosen's team realize Landy gave Bourne an address in code. Vosen races to the facility and confronts Landy, but she has already faxed away his documents. Meanwhile, Bourne is cornered on the facility's rooftop. Paz holds Bourne at gunpoint and demands to know why Bourne spared him. Bourne questions whether Paz even knows why he is supposed to kill Bourne and then says, "Look at what they make you give," repeating the dying words of the Treadstone assassin, The Professor, from The Bourne Identity. Paz lowers his weapon as Bourne moves to escape, but Vosen appears and fires on Bourne just as he jumps from the roof into the river below.
A few days later, Nicky Parsons is in hiding and watching a news report: Blackbriar is exposed, Hirsch and Vosen are arrested, and CIA Director Ezra Kramer is the subject of a Senate hearing regarding his conduct. Televised coverage of these events, and Nicky watching them, is interspersed with Bourne's sillouette motionless in the water. Finally, the TV report announced that David Webb, a.k.a. Jason Bourne, reportedly fell ten stories into the East River, but, after three days, his body was not recovered. As this is said, Nicky smiles, and the sillouette begins to swim away into the darkness.