TRAIL causes apoptosis in numerous types of tumor cells. However, the mechanisms regulating TRAIL-induced apoptosis remain to be elucidated. We have investigated the role of PKC in regulating TRAIL-induced mitochondrial events and apoptosis in the Jurkat T cell line. We found a caspase-dependent decline in mitochondrial membrane potential and translocation of cytochrome c from mitochondria into the cytosol in response to TRAIL. Both these events were prevented by PKC activation. Moreover, PKC activation considerably reduced the activation of caspases, PARP cleavage and apoptosis when induced upon TRAIL treatment. MAPK activation was involved in the mechanism of PKC-mediated inhibition of TRAIL-induced cytochrome c release from mitochondria. Furthermore, inhibition of the MAPK pathway partially reversed the PKC-mediated inhibition of TRAIL-induced apoptosis. Besides, PKC activation may also inhibit the TRAIL-induced apoptosis through a MAPK-independent mechanism. Altogether, these results indicate a negative role of PKC in the regulation of apoptotic signals generated upon TRAIL receptor activation.