Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent as it selectively kills tumor cells but spares normal cells. Resistance to TRAIL by tumor cells limits its therapeutic use. We have previously shown that protein kinase C-ε (PKCε) acts as an antiapoptotic protein in MCF-7 breast cancer cells. In the present study, we have investigated the mechanism(s) by which PKCε contributes to TRAIL resistance. Overexpression of PKCε inhibited caspase-8 and -9 activation, release of mitochondrial cytochrome c and cell death induced by TRAIL, but did not interfere with the recruitment of caspase-8 to the death-inducing signaling complex. Knockdown/inhibition of PKCε resulted in enhanced sensitivity to TRAIL. The level of Bcl-2 was increased and Bid was decreased by PKCε at both the protein and mRNA level but PKCε had no effect on Bax. Knockdown of Bcl-2 by siRNA reversed TRAIL resistance in PKCε-overexpressing cells, whereas depletion of Bid contributed to TRAIL resistance in MCF-7 cells. A decrease in Bid content was also associated with inhibition of TRAIL-induced caspase-8 activation. Furthermore, PKCε depletion or overexpression of DN-PKCε was associated with a decrease in Bcl-2 protein level. Thus, our results suggest that PKCε acts upstream of mitochondria and mediates TRAIL resistance via both Bcl-2 and Bid in MCF-7 cells.