The Film opens with a replay of the events surrounding Simon’s rescue of his sister River; in what appears to be a school setting, we see River as a child questioning the Alliance. The scene then changes to a young adult River being tested on in an Alliance facility. Simon has infiltrated the base and is able to extract River. The replay pauses. A man who is called the Operative is then shown. He has been sent to find River. He kills everyone else in the room with him.
As the Crew is in the process of raiding a bank River senses that Reavers are going to attack. Mal instructs the people inside the bank to get into the vault lock it and stay in there as long as they can and the crew manages to escape the Reavers. Simon decides that he and River are going to leave the ship the next time they dock, since it is becoming increasingly dangerous for her. As they are negotiating payment for the bounty from the bank job, River arrives and unexpectedly attacks everyone in the bar. She is about to assault Mal but Simon says the safe word which makes River stop and fall asleep. Simon and River are allowed to stay on the ship. They then contact Mr. Universe, a skilled hacker who discovers River’s outburst at the bar was caused by subliminal messaging in the advertisement that was playing at the time. The subliminal messaging is controlled by the Alliance. Mr. Universe shares that River whispered “Miranda” right before her sudden assault on the bar.
Inara, a former passenger on Serenity calls Mal. The call was a trap which is mean to lure Mal and his crew to the Operative. Mal knowing it is a trap decides to go because he thinks she may be in trouble. The Operative wants River and offers the let everyone else go if Mel turns her over. They manage to escape without having to give up River.
River has another outburst and they learn that Miranda is an uninhabitable outer planet. Serenity sets out for Haven but they discover the settlement has been attacked by the alliance. Shepherd Book (a Former Passenger) is mortally wounded. The attack was because of the Operatives search for River. The crew modifies Serenity to look like a Reaver ship, and the leave for Miranda which is located within Reaver territory. When they reach Miranda they discover it had been a normal planet, but that the streets are polluted with corpses. They find out that this was where the Reavers originated from due to a chemical designed to suppress aggression which was administered by the Alliance.
The crew asks Mr. Universe to help them get this out to the public, and he agrees. The only problem is that they crew doesn’t know that The Operative is with Mr. Universe and murdered him after the transmission ended. As serenity is exiting Reaver territory they encounter a Reaver ship. They crash into the planet. Wash is then killed by a Reaver harpoon. The surviving crew flees serenity and find Mr. Universe dead. He left a message telling them of another Transmitter. The Reavers are approaching and the crew is forced to leave. Simon is shot and River closed a door between the Reavers and the rest of the crew locking herself inside to fight the Reavers.
Mal finds the transmitter but is stopped by the Operative and they engage in battle. Mal wins against the Operative, and gets him to watch the log that reveals that the Alliance created the Reavers as he leaves to rejoin the others. As Mal is informed that River is inside with the Reapers, the door opens and we see River surrounded by dead Reavers. The Operative orders his Alliance troops to resign from their mission.
The crew buries their dead and repair Serenity. Kaylee and Simon finally have sex and the Operative informs Mal that the crew of Serenity is safe, but Mal promises to kill him if he ever sees him again. Serenity takes off, with Mal acting as pilot with the help of River.