Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) δ-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCδ to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCδ identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCδ as binding sites. In addition, the interaction between HSP25 and PKCδ induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCδ. Based on these observations, we propose that after PKCδ activation, HSP25 binds to the exposed V5 region of PKCδ. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCδ and the enhancement of HSP25 phosphorylation.