Protein kinase C δ (PKCδ) regulates DNA repair and apoptosis, and inhibition of PKCδ provides robust radioprotection. In this study, we show that depletion of PKCδ increases mitochondrial reactive oxygen species (ROS) production and induces an endogenous antioxidant response through nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in decreased basal and irradiation (IR)-induced DNA damage and cell death. Radioprotection by PKCδ depletion can be reversed with the free radical scavenger, N-acetyl-L-cysteine, indicating an essential role for the antioxidant response. Whereas mitochondrial mass and membrane potential are increased in PKCδ-depleted cells, oxidative phosphorylation and the activity of electron transport chain complex I and complex III are reduced, suggesting that electron transport chain dysfunction is the source of the increased mitochondrial ROS. The antioxidant response induced by PKCδ depletion is mediated through Sirtuin 6 (SIRT6) and Nrf2. Increased mitochondrial ROS and Nrf2 activation are reversed in PKCδ/SIRT6 double knockdown cells, indicating a central role for SIRT6 in PKCδ-regulated DNA repair and cell death. Regulation of the endogenous antioxidant state through manipulation of the PKCδ/SIRT6 signaling pathway may be a novel clinical approach for protection of healthy tissues in patients undergoing IR therapy. IMPLICATIONS: Regulation of the endogenous antioxidant state through manipulation of the PKCδ/SIRT6 signaling pathway may be a novel clinical approach for protection of healthy tissues in patients undergoing IR therapy.