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Defiance continues from where Soul Reaver 2 ended. In Soul Reaver 2, Kain saved Raziel's life by drawing the Soul Reaver from him - Raziel was meant to enter the blade and become its soul-devouring spectral half. This causes a paradox that changes time for the worse, and separates Kain and Raziel.

Kain's part of the game follows him seeking Moebius the Time Streamer, a time traveling mystic for answers to where Raziel is, how he has changed time, and later following a lead Moebius gives him to explore a Vampire citadel. During his quest, Kain finds and completes a broken talisman of Vampire construct known as the Balance Emblem. Kain discovers murals during his quest dictating an ancient war between the first races to inhabit Nosgoth, the Ancient vampires and the Hylden. After sealing the Hylden in an alternate dimension using the Pillars of Nosgoth, the Hylden cursed the Ancients with blood-thirst, sterility and immortality, turning them into the first Vampires. Kain discovers from the murals that it was prophesied that both races would have a champion arise - a Vampire champion wielding the Soul Reaver, and a Hylden champion wielding a flaming sword. Because he possesses the material Soul Reaver depicted, Kain comes to believe he is the prophesied Vampire champion. The outcome of the battle between the two champions remains ambiguous.