Following his pursuit by Kirill, Jason Bourne evades Moscow police while wounded through a train station and deals with more flashbacks of when he first joined Operation Treadstone.
Six weeks later, CIA Deputy Director Pamela Landy reveals the audiotaped confession of Ward Abbott, the late former head of Treadstone, to Director Ezra Kramer. Meanwhile, in Turin, journalist Simon Ross of The Guardian meets an informant to learn about Bourne and Operation Blackbriar, the program succeeding Treadstone. The CIA tracks Ross as he returns to London, after his mention of "Blackbriar" during a cell phone call to his editor is detected by the ECHELON system. Bourne reappears in Paris to inform Martin Kreutz, the brother of his girlfriend Marie Helena Kreutz, of her assassination in India, also in the previous film.
Bourne reads Ross's articles and arranges a meeting with him at London Waterloo station. Bourne realizes that the CIA is following Ross and helps him evade capture for a while, but when he panics and ignores Bourne's instructions, Ross is shot and killed by Blackbriar assassin Paz on orders of Deputy Director Noah Vosen. Vosen's team, reluctantly assisted by Landy, analyzes Ross's notes and identifies his source as Neal Daniels, a CIA station chief involved with Treadstone and Blackbriar. Bourne makes his way to Daniels' office in Madrid but finds it empty. He incapacitates gunmen sent by Vosen and Landy. Nicky Parsons, a former Treadstone operative who shares a history with Bourne, tells him that Daniels has fled to Tangier and aids his escape from an arriving CIA unit.
Nicky learns that Blackbriar "asset" Desh Bouksani has been tasked with killing Daniels. Vosen sees that Nicky accessed information about Daniels and sends Bouksani after Nicky and Bourne as well, a decision with which Landy fiercely disagrees. Bourne follows Bouksani to Daniels but fails to prevent Daniels's death by a planted bomb. However, Bourne manages to kill Bouksani before he can kill Nicky. Bourne opens up to Nicky and then sends Nicky into hiding. Bourne examines the contents of Daniels' briefcase and finds the address of the deep-cover CIA bureau in New York City, where Vosen directs Blackbriar. Bourne travels to New York.
Landy receives a phone call from Bourne, which is intercepted by Vosen. When Landy tells him that his real name is David Webb and gives him the birth date "4-15-71", Bourne tells Landy to "get some rest" indicating that he is in New York and watching her from an overlooking building. Vosen intercepts a text to Landy from Bourne of a location to meet up, and leaves his office with a tactical team. Bourne, however, waits for them all to leave, enters Vosen's office, and takes classified Blackbriar documents. When he realizes that he has been tricked, Vosen sends Paz after Bourne, but the resulting car chase ends with Bourne and Paz's vehicles crashing into a concrete barrier. Bourne holds the injured Paz at gunpoint, but spares his life.
Bourne arrives at a hospital at 415 East 71st Street, having figured out Landy's coded message. Outside, Bourne meets Landy and gives her the Blackbriar files before going inside. Vosen also figures out Landy's code and warns Dr. Albert Hirsch, who ran Treadstone's behavior modification program, that Bourne is coming. He follows Landy inside the building but is too late to stop her from faxing the Blackbriar documents out. Meanwhile, on an upper floor, Hirsch is confronted by Bourne, who now remembers that he volunteered for Treadstone. As Bourne flees to the roof, he is confronted by Paz, who asks, "Why didn't you take the shot?" Bourne asks Paz if he knows why he's supposed to kill him, and repeats the dying words of The Professor in The Bourne Identity: "Look at us. Look at what they make you give." Paz lowers his gun, but Vosen appears and shoots at Bourne as he jumps into the East River.
Some time later, Nicky watches a news broadcast about the exposure of Operation Blackbriar, the arrests of Hirsch and Vosen, a criminal investigation against Kramer, and the status of David Webb, a.k.a. Jason Bourne. Upon hearing that his body has not been found after a three-day search of the river, Nicky smiles. Bourne is shown swimming away in the East River.
Matt Damon as Jason Bourne, a former operative for the black ops Operation Treadstone.
Julia Stiles as Nicky Parsons, Bourne's former Treadstone contact in Paris.
David Strathairn as Noah Vosen, CIA deputy director in charge of the new Treadstone black ops upgrade called Operation Blackbriar.
Scott Glenn as Ezra Kramer, director of the CIA.
Paddy Considine as Simon Ross, a reporter for The Guardian who has been investigating Treadstone.
Édgar Ramírez as Paz, a Blackbriar assassin.
Albert Finney as Dr. Albert Hirsch, the psychologist who oversaw Treadstone's behavioral modification program.
Joan Allen as Pamela Landy, CIA deputy director and task force chief, sent in to aid Vosen in tracking down Bourne.
Daniel Brühl as Martin Kreutz, Marie's brother
Tom Gallop reprises his role as special agent Tom Cronin, Pamela Landy's assistant. Corey Johnson plays Ray Wills, Vosen's deputy at Operation Blackbriar. Joey Ansah plays Desh Bouksani, a Blackbriar asset tasked to kill Bourne in Tangier. Colin Stinton plays Neal Daniels, CIA station chief in Madrid and a former member of Treadstone, who observed David Webb's initiation into the project and his transition to Jason Bourne. Lucy Liemann plays Lucy, a Blackbriar technician. Franka Potente has an appearance in a flashback as Marie Kreutz, Bourne's murdered girlfriend.