Although activation of protein kinase C (PKC) epsilon and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKCepsilon signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKCepsilon and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKCepsilon, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKCepsilon greatly increased mitochondrial PKCepsilon expression and activity, which was concomitant with increased mitochondrial interaction of PKCepsilon with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKCepsilon activity, as the interactions were also observed in mice expressing inactive PKCepsilon. However, although both active and inactive PKCepsilon bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKCepsilon but not in mice expressing inactive PKCepsilon. Examination of potential downstream targets of mitochondrial PKCepsilon-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKCepsilon forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKCepsilon-ERK modules appears to play a role in PKCepsilon-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.