In the game's story, Valentine is the root cause of Heaven-Attained DIO's appearance in the universe, and the entire story of the game because of it. During the events of his final battle with Johnny, trapped by Tusk ACT4's power, Valentine searches the various parallel universes in hopes of something capable of saving him while he buys time against his enemy. After some searching, he encounters a version of DIO within another universe. That version of DIO was able to undo the power of the Golden Spin with only a wave of his hand, and Valentine initially looked to him for help. However, after telling the alternate DIO of the existence of parallel universes, Valentine quickly realizes his mistake: Heaven-Attained DIO immediately made it his objective to subjugate and conquer every universe possible.
With his country threatened by the so-called "terrorist", Valentine ultimately decides to ally himself with the Joestar family, as they are his best hope for defeating Heaven DIO. Appearing multiple times under the guise of being one of Heaven DIO's minions sent to fight the protagonist group, he discreetly gives aid to the Joestars through small pieces of information each meeting.
Eventually, after the Joestar family's first encounter with Heaven DIO, Valentine directly approaches them. He confirms of the original universe DIO's status and hints at the weakness of DIO's new Heaven Ascension form. While Johnny is rightfully distrustful of Valentine, Valentine attempts to diffuse his suspicion by tossing the revolver he had hidden, forcing Johnny to throw his own version as the group witnesses the menger sponge effect. Despite the evidence that Valentine bears no ill-will and has no reason to deceive the Joestars, Johnny cannot forgive him for killing Gyro.
Valentine also helps Joseph Joestar confirm the reason why the two versions of him can co-exist right next to each other: as both of them are from the same timeline and are not alternate/parallel versions of the other, they are safe from the menger sponge effect.
Eventually, Valentine's role is discovered by Heaven DIO. He reveals his reason for betrayal, following the path of true justice, even it means sacrificing his life for the sake of his country. With nowhere to run, Valentine boldly attacks DIO, but is ultimately defeated by The World Over Heaven, with all versions of him in every universe wiped from reality along with D4C.
Due to Valentine's erasure from the timelines, the ending of Steel Ball Run is altered. In the new timeline, Gyro is alive to fight THE WORLD Diego alongside Johnny.