Unapposed connexin 43 hemichannels (Cx43Hc) are present on sarcolemma of cardiomyocytes. Whereas Cx43Hc remain closed during physiological conditions, their opening under ischemic stress contributes to irreversible tissue injury and cell death. To date, conventional blockers of connexin channels act unselectively on both gap junction channels and unapposed hemichannels. Here, we test the hypothesis that Gap26, a synthetic structural mimetic peptide deriving from the first extracellular loop of Cx43 and a presumed selective blocker of Cx43Hc, confers resistance to intact rat heart against ischemia injury. Langendorff-perfused intact rat hearts were utilized. Regional ischemia was induced by 40-min occlusion of the left anterior descendent coronary and followed by 180 min of reperfusion. Gap26 was applied either 10 min before or 30 min after the initiation of ischemia. Interestingly, myocardial infarct size was reduced by 48% and 55% in hearts treated with Gap26 before or during ischemia, respectively, compared to untreated hearts. Additionally, myocardial perfusate flow was increased in both groups during reperfusion by 37% and 32%, respectively. Application of Gap26 increased survival of isolated cardiomyocytes after simulated ischemia–reperfusion by nearly twofold compared to untreated cells. On the other hand, superfusion of tsA201 cells transiently expressing Cx43 with Gap26 caused 61% inhibition of Cx43Hc-mediated currents recorded using the patch clamp technique. In summary, we demonstrate for the first time that Cx43 mimetic peptide Gap26 confers protection to intact heart against ischemia–reperfusion injury whether administered before or after the occurrence of ischemia. In addition, we provide unequivocal evidence for the inhibitory effect of Gap26 on genuine Cx43Hc.