In Chicago, Wesley Gibson works at a dead-end desk job with an overbearing boss, takes anti-anxiety medication for panic attacks, and has a live-in girlfriend who cheats on him with his best friend Barry. One night in the pharmacy, Wesley is told by a mysterious woman named Fox that his father was a recently murdered assassin, and the killer, Cross, is after him. Cross and Fox engage in a shoot-out followed by a car chase in the streets of Chicago. Wesley manages to escape and Fox then has a shoot out again with Cross, causing a car accident that knocks Wesley unconscious. Fox brings Wesley to the headquarters of The Fraternity, a thousand-year-old secret society of assassins. The group's leader, Sloan, explains that Wesley's panic attacks are actually the untrained expression of a rare superhuman ability; when stressed, the drastically increased heart rate and adrenaline levels result in bursts of superhuman strength, speed, and reflexes – demonstrated when he successfully shoots the wings off of a fly. The Fraternity can teach him to control this ability, so Wesley can follow in his father's footsteps as an assassin, beginning by inheriting his fortune. Wesley is initially reluctant and returns to work, only to finally snap when discovering several million dollars in his bank account. He excoriates his boss in front of the entire office, and on his way out, smashes Barry in the face with a computer keyboard. Fox is waiting outside to take him back to the Fraternity headquarters—an unassuming textile mill.
Wesley is then subjected to brutal training; among other forms of combat, he learns to fire bullets to curve around objects. Afterward, Wesley is shown the Loom of Fate, a loom that gives the names of the targets through binary code hidden in weaving errors of the fabric. Those the Loom identifies will apparently cause tragedy in the future; but only Sloan sees and interprets the names fate wishes to see dead. Wesley is initially reluctant about killing people. Then Fox reveals that in her childhood, a hired killer burned her father alive in front of her—and said hitman was supposed to be killed by the Fraternity before that, but the assassin failed to pull the trigger. She now considers preventing such tragedy her mission.
After several routine missions and a chance meeting with Cross, in which Wesley is shot in the arm with a deliberately traceable bullet, Sloan grants Wesley's wish to avenge his father and sends him after Cross—but then secretly gives Fox a mission to kill Wesley, saying that his name had come up in the Loom as well. Analyzing the bullet that hit Wesley, it is discovered that the manufacturer was Pekwarsky, a bullet-maker living in eastern Moravia, the birthplace of the Fraternity. Wesley and Fox travel there and capture Pekwarsky, who arranges a meeting with Cross. Wesley faces Cross alone on a moving train. Fox steals a car and crashes it into the train, eventually causing a derailment. After Cross saves Wesley's life by preventing him from falling into a ravine, Wesley fatally shoots him. Before dying, Cross reveals that he is Wesley's real father. Fox confirms this, and explains that Wesley was recruited because he was the only person that Cross would not kill. Fox then reveals the kill order on Wesley and raises her gun, but Wesley escapes by shooting out the glass underneath him and plunging into the river below.
Wesley is retrieved by Pekwarsky, who takes him to his father's apartment, located across the street from Wesley's old home. Pekwarsky explains that Sloan started manufacturing targets for profit after discovering that he was targeted by the Loom of Fate, and did not tell the Fraternity members that they were now nothing more than paid killers. Cross discovered the truth and went rogue, and started killing Fraternity members to keep them away from his son. Pekwarsky departs, stating that Wesley's father wished him a life free of violence. Wesley, however, decides to take out Sloan after discovering a secret room containing all of his father's weapons and maps.
An enraged Wesley assaults the Fraternity's textile mill-fortress and battles his way through it, killing nearly every Fraternity member in the process. Upon entering Sloan's office, he reveals Sloan's deception to the master assassins present in the room. Sloan reveals that all of their names had come up in the weaving, and that he had merely acted to protect them. Were they to follow the code, every one of them should kill themselves on the spot. Fox, who believes in the code more than anyone due to her own experience, turns on her fellow assassins, and curves a bullet that kills every Fraternity member in the room, including herself, but not before she throws her gun to Wesley to distract him from trying to save Fox. Sloan manages to escape, and Wesley destroys the Loom of Fate.
Wesley, penniless once again, is left aimless. A man is then seen at a computer, much like Wesley at the beginning of the film. Sloan appears and points a gun at the back of the man's head. At that moment, the man turns around and is revealed to be a decoy. Sloan is then killed by Wesley using a long-distance bullet. Similar to the comic book miniseries, the film ends with Wesley stating his accomplishments, then turning to the camera, breaking the fourth wall and asking the audience "What the fuck have you done lately?", then the credits roll.