The Herta Space Station is attacked by members of the Antimatter Legion, followers of the Path of Destruction. Amidst the chaos, two Stellaron Hunters, Kafka and Silver Wolf, infiltrate the station and steal the Stellaron that was being safely held within it. Kafka takes hold of the Stellaron and inserts it into the body of an empty vessel generated by Silver Wolf (whose gender and name are chosen by the player). The vessel (known as the Trailblazer in English and various other names in other translations of the game) awakens, after which Kafka erases their memory, informs them of the future that lies ahead, and leaves. They then fall unconscious.
A now-amnesic Trailblazer wakes up and meets March 7th and Dan Heng. Both are traveling on the Astral Express, a large cosmic train that travels the universe along the eponymous "Star Rail". After eliminating the remaining threats from the Antimatter Legion, the Trailblazer meets Himeko, the Navigator of the Astral Express, and Welt Yang, an interstellar traveler originating from the world of Honkai Impact 3rd. In an attempt to discover their origins and eliminate the threats of the remaining Stellarons, the Trailblazer departs with the Astral Express crew.
