In a flashback, the Bride (Uma Thurman) — visibly pregnant — and her groom (Chris Nelson) rehearse their wedding. Bill (David Carradine), her former lover and the leader of the Deadly Viper Assassination Squad, arrives unexpectedly and wishes her well, and it is revealed that the Bride had retired from assassination and left Bill in order to give a better life to her unborn daughter. Moments later, the other assassination squad members arrive and attack the wedding rehearsal on Bill's orders.
In the present, Bill warns his brother Budd (Michael Madsen), a former Deadly Viper and now a bouncer, that he will be targeted next. The Bride arrives at his trailer and bursts through the door, expecting to ambush him, but Budd is expecting her, shooting her in the chest with a double-barreled shotgun blast of rock salt, then sedates her. Budd calls Elle Driver (Daryl Hannah), another former Deadly Viper, offering to sell her the Bride's Hanzō sword for a million dollars cash. He then seals the Bride inside a coffin and buries her alive.
A flashback shows Bill taking the Bride to be trained by legendary martial arts master Pai Mei (Gordon Liu). She eventually gains his respect and learns a number of techniques, including the art of punching through thick planks of wood from inches away, and a technique, taught to nobody else, of killing using apparently non-lethal touches to pressure points. She uses the former skill to break out of the coffin and claws her way to the surface.
Elle arrives at Budd's trailer for their transaction but has hidden a lethal black mamba with her money, which kills him. She then tells Bill (by phone) that the Bride has killed Budd by putting a black mamba (the deadly viper that was the Bride's assassination squad code name) in his trailer, and that Elle has killed the Bride. As she exits the trailer, she is ambushed by the Bride, who had arrived there soon after Elle. In the middle of an all-out battle in the trailer, Elle taunts the Bride with the news that Elle had poisoned Pai Mei in revenge for his snatching out her eye when she called him a 'miserable old fool'. The Bride then plucks out Elle's remaining eye and leaves her screaming and thrashing about in the trailer with the black mamba.
The Bride travels to Mexico to query an elderly gentlemen about Bill's whereabouts. The man, who is the proprietor of a brothel, was purportedly a father figure to a younger Bill. Regardless, the man divulges Bill's location.
After finding Bill deep in the Mexican countryside, the Bride is shocked to find her now-four-year-old daughter B.B. (Perla Haney-Jardine) alive and well. She spends the evening with Bill and B.B. After B.B. has gone to bed, Bill shoots the Bride with a dart containing a truth serum and interrogates her. A flashback recalls the Bride's discovery of her pregnancy while on an assassination mission, and her resulting decision to call off her assignment and leave the Deadly Vipers. The Bride explains that she ran away without telling Bill in order to protect their unborn daughter from him and his life. Though Bill understands, he remains unapologetic for what he did, explaining that he's a murdering bastard and there are consequences to breaking the heart of a murdering bastard. They fight, but although the Bride loses her weapon, she disables Bill with Pai Mei's fatal pressure point technique, which he had secretly taught her. Bill, aware of the technique and that he will shortly die, makes his peace with the Bride and dies. The Bride departs with B.B.; later they are seen watching cartoons in a hotel together after Beatrix gives thanks.