Five hundred years in the future, mankind has abandoned Earth and emigrated to a neighboring star system. In the place of national governments, the interplanetary Alliance wields tight political power over inhabitants of the wealthy "central planets", while life on the partially-colonized "Outer Rim" planets resembles the lawlessness and desolation of the American Old West. In the depths of an Alliance facility, Simon Tam rescues his sister River Tam from her scientist captors, who experiment on her with hopes of harnessing her psychic abilities as a weapon. In hiding from the Alliance, the siblings eventually find safe harbor on the spacecraft Serenity, filled with a ragtag crew that makes a living by taking jobs of varying legality in the Outer Rim.

After a bank heist on an Outer Rim planet is disrupted by a horde of feral, spacefaring cannibals known as Reavers, the crew narrowly escapes back to Serenity. Simon accosts the ship's captain, Malcolm "Mal" Reynolds, protesting River's involvement in the raid and her exposure to danger. He declares that he and River will leave Serenity at the next spaceport.

The crew disembarks at a trading post and enters a bar to meet with Fanty and Mingo, the men who hired Serenity for the robbery. River wanders into the bar and observes a television advertisement, which causes her to immediately and brutally attack the other patrons until Simon arrives and utters a code phrase, which renders River unconscious. Thoroughly confused, Mal takes River and Simon back to Serenity, where Simon reveals that River was conditioned to be an Alliance assassin. Mal contacts Mr. Universe, a reclusive techno-geek who analyzes the bar security camera footage and discovers that a subliminal message in the advertisement triggered River's attack. Unknown to the crew, the message was placed by the Operative, a self-described monster with no name or rank, assigned by the Alliance to retrieve River at all costs.

Mal receives a call from Inara Serra, a former passenger, asking for help with local unrest. Recognizing that he is entering a trap, Mal visits Inara and finally meets the Operative, who offers to release Mal if he surrenders River. Mal refuses and, following a vicious struggle with the Operative, escapes with Inara to Serenity. Aboard the ship, River reveals the existence of "Miranda," an Outer Rim planet deemed uninhabitable by the Alliance and located on the far side of a Reaver fleet. The crew discovers that the Operative has attacked all of Serenity's ports of refuge, killing their former shipmate Shepherd Book in the process. Stricken with guilt and vengeance, Mal overrides his crew's protests and disguises Serenity as a Reaver vessel, allowing them to sneak through the Reaver fleet unmolested and land on Miranda, a planet that contains only corpse-filled cities despite being fully habitable. A holographic recording from Dr. Caron, a regretful Alliance scientist, explains that the Alliance attempted to bring peace to the population by filling the atmosphere with a drug designed to suppress aggression. The drug instead suppressed the people's motivation to do anything and they died of starvation, while 0.1% had the opposite reaction and became the hyper-violent Reavers. Mal declares his intentions to broadcast this message to the solar system via Mr. Universe's powerful transmitters, aware of the Operative and the certain trap that awaits them.

As the Operative mobilizes an Alliance fleet above Mr. Universe's station, Mal provokes the Reaver fleet into pursuing Serenity and leads it into a massive space battle with the Alliance. With both sides distracted, Serenity, pursued by a Reaver ship and the Operative in an escape pod, crash-lands on the station, where they are surprised by Reavers who kill Wash, the pilot. The crew enters the station and sets up defense positions, while Mal descends to transmit the Miranda message. As the crew's defense crumples beneath the Reaver assault, they retreat behind a blast door, which jams before it can close. With everyone wounded and ammunition low, River dives through the blast door and seals it. In the depths of the station, Mal is attacked by the Operative, whom he narrowly defeats and incapacitates, leaving the man to watch the broadcast of the Miranda message. As a wounded and exhausted Mal rejoins the crew, the blast door opens, revealing River standing victorious amid piles of dead Reavers. Alliance troops burst onto the scene, but the Operative, his faith in the Alliance shattered by the Miranda message, orders the soldiers to stand down.

The crew buries their deceased friends and repairs Serenity. As the ship prepares to leave, the Operative approaches Mal and warns him of possible retaliation by the Alliance. Mal returns to Serenity's cockpit and with his new pilot River, blasts into space.