Although functional coupling between protein kinase Cε (PKCε) and mitochondria has been implicated in the genesis of cardioprotection, the signal transduction mechanisms that enable this link and the identities of the mitochondrial proteins modulated by PKCε remain unknown. Based on recent evidence that the mitochondrial permeability transition pore may be involved in ischemia/reperfusion injury, we hypothesized that protein-protein interactions between PKCε and mitochondrial pore components may serve as a signaling mechanism to modulate pore function and thus engender cardioprotection. Coimmunoprecipitation and GST-based affinity pull-down from mouse cardiac mitochondria revealed interaction of PKCε with components of the pore, namely voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT), and hexokinase II (HKII). VDAC1, ANT1, and HKII were present in the PKCε complex at ≈2%, ≈0.2%, and ≈1% of their total expression, respectively. Moreover, in vitro studies demonstrated that PKCε can directly bind and phosphorylate VDAC1. Incubation of isolated cardiac mitochondria with recombinant PKCε resulted in a significant inhibition of Ca²⁺-induced mitochondrial swelling, an index of pore opening. Furthermore, cardiac-specific expression of active PKCε in mice, which is cardioprotective, greatly increased interaction of PKCε with the pore components and inhibited Ca²⁺-induced pore opening. In contrast, cardiac expression of kinase-inactive PKCε did not affect pore opening. Finally, administration of the pore opener atractyloside significantly attenuated the infarct-sparing effect of PKCε transgenesis. Collectively, these data demonstrate that PKCε forms physical interactions with components of the cardiac mitochondrial pore. This in turn inhibits the pathological function of the pore and contributes to PKCε-induced cardioprotection.