Two years after the events of the first film, Jason Bourne and Marie Kreutz are now in Goa, India. Still experiencing flashbacks about his former life as a CIA assassin, he records them in a notebook.
In Berlin, CIA agents subordinate to Deputy Director Pamela Landy are paying US$3 million for the "Neski files", documents on the theft of $20 million in allocation money seven years prior. The deal is interrupted by Kirill, an agent for Russia's Federal Security Service who works for Russian oil oligarch Yuri Gretkov. Kirill plants Bourne's fingerprint at the scene, interrupts the deal, kills everyone involved and steals the files and money. He travels to Goa to kill Bourne, but Bourne spots him and flees with Marie. Mistakenly believing Bourne is the driver, Kirill shoots at the driver seat and kills Marie. Their vehicle plunges into a river and Bourne leaves after unsuccessfully attempting to revive Marie.
Bourne travels to Naples, Italy with money and passports. After finding Bourne's fingerprint that Kirill planted, Landy asks Deputy Director Ward Abbott about Operation Treadstone, the defunct CIA program to which Bourne belonged. She tells Abbott that the CIA agent who stole the $20 million was named in the Neski files. Some years previously, Russian politician Vladimir Neski was about to identify the thief when he was supposedly murdered by his wife in a Berlin hotel. Landy believes that Bourne and Treadstone's late supervisor, Alexander Conklin, were somehow involved. She also believes that Bourne killed her two agents. Both Abbott and Landy go to Berlin to capture Bourne.
In Naples, Bourne allows himself to be identified by security. He subdues his CIA interrogator and copies the SIM card from his cell phone. From the subsequent phone call, he learns about Landy and his frame up. Bourne goes to Munich to visit Jarda, the only other remaining Treadstone operative. Jarda informs Bourne that Treadstone was shut down after Conklin's death, and tries to incapacitate him. Bourne kills Jarda and escapes before the CIA arrives. Bourne follows Landy and Abbott as they meet former Treadstone support technician Nicky Parsons to question her about Bourne. Believing that the CIA is hunting him again, Bourne calls Landy from a nearby roof and is told of the reason for his pursuit. He demands a meet-up with Nicky and indicates to Landy that he can see her in the office, shocking the entire CIA team.
Bourne kidnaps Nicky at the meet-up in Alexanderplatz, and learns from her that Abbott was the head of Treadstone, not Conklin. He remembers that he murdered Neski in Berlin, but Nicky knows nothing about it, so he lets her go. Bourne then visits the hotel where the killing took place and recalls more of his mission?he killed Neski on Conklin's orders, and when Neski's wife showed up, he shot her to make it look like a murder?suicide. When Danny Zorn (Conklin's assistant) suspects a conspiracy against Bourne, he confides in Abbott, who kills him. Bourne breaks into Abbott's hotel room and records a conversation between him and Gretkov that incriminates them in the theft of the 20 million. Abbott admits to Bourne that he stole the money, ordered Bourne to murder Neski, ordered Kirill to retrieve the files, and have Bourne framed and silenced in Goa. After Bourne leaves, Landy confronts Abbott about her suspicion and he commits suicide. Bourne sends the tape of the confession to Landy, vindicating himself.
Bourne goes to Moscow to find Irena Neski, the daughter of Vladimir Neski. Kirill, tasked once again by Gretkov with killing Bourne, finds and wounds him. Bourne flees on a stolen taxi and Kirill chases him. After a long high-speed chase also involving police vehicles, Bourne forces Kirill's vehicle into a concrete divider. Bourne walks away, leaving a mortally wounded Kirill. Gretkov is subsequently arrested. Bourne locates Irena, confesses to murdering her parents and apologizes to her, then leaves. Later in New York, Bourne calls Landy; she thanks him for the tape and also asks him to meet her. Bourne tells her to take a rest. Landy then reveals to Bourne his original name and tells him his date of birth (which is actually a code).