Protein kinase Cε (PKCε) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCε colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 ± 3.4% vs. 23.7 ± 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 ± 6.0% vs. 25.1 ± 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 ± 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.