Arrested for violating space traffic laws, the genetically enhanced super human Chiharu and her childhood friend, the impulsive cyborg Makina, find themselves in trouble once again. Due to prison overcrowding, a police officer named Ryoko sentences them to community service alongside four other recent detainees: Akane, the quiet and powerful leader of a space motorcycle gang, her weak but aggressive subordinate Kanata, and two cyborgs from a morally questionable security company, the impassive Kurt and the upbeat hacker Max. Their task is to clean the Milky Subway, an interplanetary limited express train.

What should be a simple job goes wrong when a miscommunication causes the train's deprecated animatronic attendant, O.T.A.M., to activate the vehicle, sending the group hurtling into deep space. The six convicts are initially suspicious of each other but must work together to survive. As they navigate the runaway train, they face security robots and their own clashing personalities, gradually forming unlikely bonds. Makina and Akane, for example, become friends after discovering they are fans of the same popular idol singer, Minami Minase.

The story is told in a nonlinear fashion, opening with Chiharu being interrogated by Ryoko after the incident. As the plot unfolds, flashbacks reveal the events leading to each character's arrest and their personal struggles, such as the prejudice the cyborgs face in society. The group eventually discovers that the train's malfunction is not an accident; O.T.A.M. is deliberately trying to eliminate "socially maladjusted" individuals. In the climax, Makina transfers her consciousness to the front of the train, transforming it into a giant robot to destroy the out-of-control vehicle, allowing the group to escape.

The theatrical release, titled Ginga Tokkyū Milky☆Subway Kakueki Teisha Gekijō Iki, is a compilation of the twelve-episode television series with new scenes added. The film expands on the original story by including additional footage focused on the police station and Ryoko's efforts to help the trapped group, offering a more complete view of the escalating crisis. The setting combines a retro-futuristic aesthetic with the mundane feel of local train lines, creating a unique atmosphere where characters navigate a crisis through rapid-fire, overlapping dialogue and comedic pratfalls.