Both glycogen synthase kinase 3β (GSK3β) and the ATP-dependant potassium channel (K_ATP) mediate opioid-induced cardioprotection (OIC). However, whether direct K_ATP channel openers induce cardioprotection prior to reperfusion and their signaling cascade position with respect to GSK3β inhibition is unknown. Therefore, we investigated the role of K_ATP channel opening at reperfusion in OIC, and the interaction between the GSK signaling axis and K_ATP channels in cardioprotection.Male Sprague-Dawley rats underwent 30 minutes ischemia with 2 hours of reperfusion and infarct size was determined. Rats given the nonselective opioid agonist, morphine (0.3 mg/kg), or the selective delta opioid agonist, BW373U86 (1.0 mg/kg), 5 minutes prior to reperfusion reduced infarct size (40.3±1.6*, 39.7±1.9* versus 60.0±1.1%, respectively, * P<0.001%). This protection was abrogated with prior administration of the putative sarcolemmal K_ATP antagonist, HMR-1098 (6 mg/kg), or the putative mitochondrial K_ATP antagonist, 5-HD (10 mg/kg). The putative sK_ATP channel opener, P-1075 (1μg/kg) or the putative mK_ATP channel opener, BMS-191095 (1 mg/kg) given 5 minutes prior to reperfusion also reduced infarct size (41.8±2.4*, 43.4±1.4*) and protection was abrogated by prior administration of the PI3k inhibitor wortmannin (60.0±1.7, 64.0±2.6%, respectively, * P<0.001). Cardioprotection afforded by the GSK inhibitor SB216763 (0.6 mg/kg) given 5 minutes prior to reperfusion was also partially blocked by either HMR or 5-HD and completely blocked when HMR and 5-HD were given in combination (40.8±1.6*, 50.4±1.6^; 49.4±1.7^, 61.6±1.6%, respectively, * or ^ P<0.001). These data indicate that both the sK_ATP and mK_ATP channel are involved in acute OIC and the GSK signaling axis regulates cardioprotection via K_ATP channel opening.