In the intrinsic pathway of apoptosis, mitochondria play a crucial role by releasing cytochrome c from the inter-membrane space into the cytoplasm. Cytochrome c release through Bax/Bak-dependent channels in mitochondria has been well-documented. In contrast, cyclophilin D (CypD), an important component of permeability transition (PT) pore -dependent protein release remains largely undefined, and no apoptogenic proteins that act specifically in a CypD-dependent manner have been reported to date. Here, we describe a novel and evolutionarily conserved protein, Apoptogenic Protein (Apop). Mouse Apop-1 expression induces apoptotic death by releasing cytochrome c from mitochondria into the cytosolic space, followed by activation of caspase-9 and -3. Apop-1-induced apoptosis is not blocked by Bcl-2 or Bcl-xL, inhibitors of Bax/Bak-dependent channels, while it is completely blocked by cyclosporin A, an inhibitor of PT pore. Cells lacking CypD were resistant to Apop-induced apoptosis. Moreover, inhibition of Apop expression prevented the cell death induced by apoptosis-inducing substances. Our findings thus indicate that the expression of Apop-1 induces apoptosis though CypD-dependent pathway, and that Apop-1 plays roles in cell death under physiological conditions.