Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.