Using two-dimensional electrophoresis, mass spectrometry, immunoblotting, and affinity pull-down assays, we found that myocardial protein kinase C ε (PKCε) is physically associated with at least 36 known proteins that are organized into structural proteins, signaling molecules, and stress-responsive proteins. Furthermore, we found that the cardioprotection induced by activation of PKCε is coupled with dynamic modulation and recruitment of PKCε-associated proteins. The results suggest heretofore-unrecognized functions of PKCε and provide an integrated framework for the understanding of PKCε-dependent signaling architecture and cardioprotection.