Following the events of Legacy of Kain: Soul Reaver 2, Raziel and Kain were trapped in the Serafan fortress. Separated by sudden events, Raziel was forced to retreat into the spectral realm, leaving Kain to escape from the Serafan on his own. Both must escape the predicament they find themselves in and fight back against those who would seek to use them as manipulations in a grander plot. From both heroes cry out shouts of defiance, and a thirst to find the revelations that have been buried in the world of Nosgoth.

Legacy of Kain: Defiance is a third-person action game with light puzzle-solving elements, combining concepts seen in both Blood Omen 2 and Soul Reaver 2. The story unfolds through chapters, focusing alternately on Kain and Raziel as they pursue their separate, though intertwined quests. Control of each character alternates between levels, however both are controlled from a behind-the-shoulder perspective. While both heroes are different, they are similar in movement, ability to attack, defend, jump and access special abilities.

Kain and Raziel no longer pick up weapons, instead both using a form of the Soul Reaver weapon (Kain uses the physical sword form, Raziel uses an energy blade). Each Soul Reaver can now be "charged" to unleash a special ability, determined by the current alignment of Soul Reaver at the time. Both Kain and Raziel lose energy at a constant rate and must feed to replenish life energy (Kain feeds on the blood of living creatures, Raziel feeds on the souls of living and non-living creatures). And both Kain and Raziel feature telekinetic abilities to manipulate objects at a distance.

The two are, however, different characters that play differently, each requiring use of his talents, to bypass obstacles (sometimes the same obstacle). Kain focuses on strong combat and destructive abilities. He is able able to power up the Soul Reaver weapon using properties of the Pillars of Nosgoth. Raziel is more subtle, able to use stealth and the phasing between material and spectral worlds in order to discover new paths. As in Soul Reaver, Raziel can phase into the spectral world at any time but must find a special location in order to phase back. Raziel powers up the Soul Reaver using elemental powers.