In the 26th century humanity has colonized a new solar system. The central planets formed the Alliance and won a war against the outer planet Independents who resisted joining the Alliance. River Tam is conditioned by Alliance scientists into becoming a psychic and an assassin. She is rescued by her brother Dr. Simon Tam. During her training, River inadvertently read the minds of several top government officials and learned their secrets. Consequently, an Alliance agent known only as the Operative is tasked with recapturing her.
The siblings have found refuge aboard the transport spaceship Serenity with Captain Malcolm “Mal” Reynolds, first mate Zoe Washburne, pilot Hoban “Wash” Washburne, mercenary Jayne Cobb, and mechanic Kaylee Frye. Despite Simon’s objections, Mal brings River on a bank robbery. River warns them that savage and cannibalistic Reavers are coming. They escape, but Simon decides he and River will leave Serenity at the next port. Once there, however, a subliminal message in a television commercial causes River to attack numerous bar patrons, and Mal takes the siblings back aboard the ship. The crew contacts reclusive hacker Mr. Universe, who discovers the message designed to trigger River’s mental conditioning. He notes River whispered “Miranda” before attacking and warns that someone else has viewed the footage.
Mal receives an invitation from Inara Serra. Realising it is a trap, Mal goes to confront the Operative who promises to let him go free if he hands over River. Mal barely escapes. Miranda is discovered to be a planet located beyond a region of space swarming with Reavers. The crew flies to the planet Haven but find it devastated and their friend Shepherd Book mortally wounded. The Operative promises to kill anyone who assists them until he gets River.
Mal has the crew disguise Serenity as a Reaver ship and they travel to Miranda undetected. They find all its colonists dead, and a recording that explains an experimental chemical to suppress aggression had been added into Miranda’s atmosphere. The population became so docile they stopped performing all activities of daily living and placidly died. A small proportion of them had the opposite reaction and became insanely aggressive and violent. The Alliance had created the Reavers and this was the secret in River’s subconscious.
Mr. Universe agrees to broadcast the recording, however the Operative kills him and prepares an ambush. Knowing this, the crew provoke the Reaver fleet to chase them into to the Alliance armada. The Reavers and Alliance battle while Wash pilots Serenity through the crossfire. He crash lands near the broadcast tower before being killed by a Reaver spear.
The crew make a last stand against the Reavers to buy Mal time to broadcast the recording. The crew retreats behind a set of blast doors that fail to properly close. Simon is shot, and River dives through the doors to throw back Simon’s medical kit and close the doors before the Reavers drag her away. At the transmitter Mal fights the Operative, finally subduing him, and forces him to watch as the recording is broadcast. Mal returns to the crew and the blast doors open to reveal that River has killed all the Reavers. The Operative orders the Alliance troops to stand down.
The Operative provides medical aid and resources to repair Serenity. He tells Mal the broadcast has weakened the Alliance government, but while he will try to convince the Parliament that River and Simon are no longer threats, he warns that they may continue their pursuit in retribution for getting the word out. Serenity takes off with River as Mal’s new pilot.