Ischemic preconditioning (PC) suppresses chemical coupling of cardiomyocytes via gap junctions (GJs) during ischemia, which is an adjunct mechanism of protection. The aim of this study was to characterize roles of protein kinases in PC-induced GJ modulation. In isolated rat hearts, ventricular tissues were sampled before and after ischemia with or without PC, and intercalated disc-rich fractions were separated for immunoprecipitation and immunoblotting. Levels of protein kinase C (PKC)-ε, p38mitogen-activated protein kinase (MAPK)-α, and Src coimmunoprecipitated with connexin-43 (Cx43) were increased after ischemia, whereas p38MAPKβ was not detected in the Cx43 immunoprecipitates. PC did not modify the level of Cx43-Src complex after ischemia. However, PC enhanced Cx43-PKCε complex formation, which was abolished by PKCε translocation inhibitory peptide (TIP). In contrast, PC reduced Cx43-p38MAPKα complex level and p38MAPK activity in the Cx43 immunoprecipitates after ischemia. The effect of PC on Cx43-p38MAPKα interaction was mimicked by SB-203580, a p38MAPK inhibitor. PC reduced permeability of GJs to Lucifer yellow in the myocardium at 25 min after ischemia, and this effect was abolished by PKCε-TIP. SB-203580 increased the GJ permeability at 15 min after ischemia compared with that in untreated controls, but the difference became insignificant 25 min after ischemia. In conclusion, PC has distinct effects on interaction of GJ Cx43 with PKCε, p38MAPKα, and Src during ischemia. Suppression of GJ permeability during ischemia by PC is primarily achieved by enhanced interaction of Cx43 with PKCε, which overwhelms the counterbalancing effect of reduced Cx43-p38MAPKα interaction.