Introduction: Ischemia/Reperfusion (IR) injury mainly causes the increase of enzymes involved in myocytes injury including CK-MB (creatine kinase-MB) isoenzyme and LDH (lactate dehydrogenase). Leakage of CK-MB isoenzyme and LDH from myocardial tissues to blood is indicator of acute myocardial infarction. The aim of this study was to assess the effect of HEMADO on IR injury and its relationship with mitochondrial ATP-sensitive K+ channels (mitoKATP) in rat heart. Methods: Twenty eight male Wistar rats (250-300g) were divided into four groups (seven members in each group): control (without ischemia), I/R (with ischemia+without HEMADO), ischemia received HEMADO (HEMADO), ischemia received HEMADO and 5-HD (5-hydroxydecanoate, specific mitoKATP channel blocker) (HEMADO+5-HD). The animals were anesthetized and the hearts were quickly removed and mounted on Langendorff apparatus and perfused by Krebs-Henseleit solution under constant pressure and temperature of 37ºC. After 20 minutes of stabilization, ischemic groups were exposed to 40 minutes of global ischemia and consecutive 90 minutes of reperfusion. Results: IR injury increased the level of LDH and CK-MB in the collected coronary flow during 5 minutes since start of reperfusion. HEMADO reduced the enzymes’ levels and using 5-HD abolished the effect of HEMADO. Conclusion: Our findings indicated that HEMADO could protect the heart against ischemia-reperfusion injury by decreasing the CK-MB and LDH levels. The cardioprotective effect of HEMADO may be mediated in part by mitoKATP.