We have previously shown that protein kinase Cε (PKCε) protects breast cancer cells from tumor necrosis factor-α (TNF)-induced cell death. In the present study, we have investigated if the antiapoptotic function of PKCε is mediated via Akt and the mechanism by which PKCε regulates Akt activity. TNF caused a transient increase in Akt phosphorylation at Ser 473 in MCF-7 cells. Overexpression of PKCε in MCF-7 cells increased TNF-induced Akt phosphorylation at Ser473 resulting in its activation. Knockdown of PKCε by small interfering RNA (siRNA) decreased TNF-induced Akt phosphorylation/activation and increased cell death. Introduction of constitutively active Akt protected breast cancer MCF-7 cells from TNF-mediated cell death and partially restored cell survival in PKCε-depleted cells. Depletion of Akt in MCF-7 cells abolished the antiapoptotic effect of PKCε on TNF-mediated cell death. Akt was constitutively associated with PKCε and DNA-dependent protein kinase (DNA-PK), and this association was increased by TNF treatment. Overexpression of PKCε enhanced the interaction between Akt and DNA-PK. Knockdown of DNA-PK by siRNA inhibited TNF-induced Akt phosphorylation and the antiapoptotic effect of Akt and PKCε. These results suggest that PKCε activates Akt via DNA-PK to mediate its antiapoptotic function. Furthermore, we report for the first time that DNA-PK can regulate receptor-initiated apoptosis via Akt.