Protein kinase Cε (PKCε) acts as an antiapoptotic protein and inhibits tumor necrosis factor-α (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in MCF-7 breast cancer cells. Members of the TNF receptor superfamily trigger apoptosis independent of the tumor suppressor protein p53, which primarily affects DNA damage-induced apoptosis. We have previously shown that PKCε acts upstream of Akt to inhibit receptor-initiated cell death. Since Akt can regulate p53, we have examined the involvement of p53 in PKCε-mediated TRAIL resistance. Overexpression of PKCε in MCF-7 cells (MCF-7/PKCε) caused a decrease in p53 and an increase in human homolog of murine double minute 2 (Hdm2) and phospho-Hdm2. Depletion of p53 by siRNA attenuated, whereas depletion of Hdm2 enhanced TRAIL-mediated apoptosis. Knockdown of Akt decreased Hdm2 phosphorylation, increased p53 level and potentiated TRAIL-induced cell death. Depletion of PKCε from MCF-7 cells caused an increase in p53, whereas knockdown of p53 caused a decrease in Bid mRNA. Depletion of Akt from MCF-7/PKCε cells resulted in an increase in p53 and Bid. These results suggest that PKCε mediates TRAIL resistance by Akt-mediated phosphorylation of Hdm2 resulting in suppression of p53 expression and downregulation of Bid in MCF-7 breast cancer cells.