This revealing real-life saga centres around a young, starstruck Robert Ford (Casey Affleck), who naively seeks out Jesse James (Brad Pitt), when the James gang is planning a train robbery in Blue Cut, Missouri, making unsuccessful attempts to join the gang with the help of his older brother Charley (Sam Rockwell), already a member.
The train turns out to be carrying only a fraction of the money originally thought, and Frank James (Sam Shepard) tells Charley Ford that this robbery would be the last the James brothers would commit, and that the gang "gave up their nightridin' for good." Jesse returns home to Kansas City, bringing the Fords, Dick Liddil (Paul Schneider) and his cousin, Wood Hite (Jeremy Renner). Jesse sends Charley, Wood and Dick away, but insists that Bob stay. He wanted the younger man just for his help in moving furniture to a new home in St. Joseph. Bob becomes more admiring of James before being sent back to his sister's farmhouse, where he rejoins his brother Charley, Hite, and Liddil.
Liddil reveals to Bob that he is in collusion with another member of the James gang, Jim Cummins, to capture Jesse for a substantial bounty. Meanwhile, Jesse visits another gang member, Ed Miller (Garret Dillahunt), who gives away information on Cummins' plot. Jesse kills Miller, then departs with Liddil to hunt down Cummins. Unable to locate him, Jesse viciously beats Albert Ford (Jesse Frechette), a young cousin of Bob and Charley. Liddil returns to the Boltons' farmhouse, and argues with Hite, which ends with Bob Ford killing Hite. They dump his body in the woods to conceal the murder from Jesse.
Jesse and Charley Ford travel to St. Joseph, Missouri, where Jesse learns of Hite's disappearance, which Charley denies knowing anything about. Meanwhile, Bob goes to Kansas City police commissioner, Henry Craig (Michael Parks), saying he knows about Jesse James' whereabouts. To prove his allegiance with the James gang, Bob urges Craig to arrest Dick Liddil. Following Liddil's arrest and confession to participation in numerous gang robberies, Bob brokers a deal with the Governor of Missouri, Thomas T. Crittenden (James Carville). He is given 10 days to capture or kill Jesse James, and promised a substantial bounty and full pardon for murder.
Charley persuades Jesse to take Bob Ford into the gang; the brothers travel to Jesse's home in St. Joseph. Introduced as cousins to the Howards (the James' pseudonym), they stay with the family, including Zee James (Mary-Louise Parker) and their two children. Jesse wants to revive his gang by robberies with the Fords, beginning with the Platte City bank.
On the morning of April 3, 1882, Jesse and the Ford brothers prepare to depart for the Platte City robbery. Jesse reads about the arrest and confessions of Liddil in the morning paper. While the three men are in the living room, Jesse removes his gun belt, and climbs a chair to clean a dusty picture. Robert Ford shoots him in the back of the head, and the Ford brothers flee. They send a telegram to the governor to announce Jesse's death, for which they were to receive $10,000. They never receive more than $500 each.
After the murder, the Fords become celebrities for a time, touring with a theater show in Manhattan, in which they re-enact the killing, but people dislike that Ford shot James in the back. Guilt-stricken, Charley writes numerous letters to Zee James asking for her forgiveness, but does not send them. Suffering from terminal tuberculosis, he commits suicide in May 1884. Bob works around the West. On June 8, 1892, he is murdered by Edward O'Kelley (Michael Copeman), at his saloon in Creede, Colorado. O'Kelley is sentenced to life in prison, but Colorado Governor James Bradley Orman pardons him after ten years in 1902.