This study tested the hypothesis that inhibition of myocardial injury and modulation of mitochondrial dysfunction by postconditioning (Postcon) after 24 h of reperfusion is associated with activation of K_ATP channels. Thirty dogs undergoing 60 min of ischemia and 24 h of reperfusion (R) were randomly divided into four groups: Control: no intervention at R; Postcon: three cycles of 30 s R alternating with 30 s re-occlusion were applied at R; 5-hydroxydecanoate (5-HD): the mitochondrial K_ATP channel blocker was infused 5 min before Postcon; HMR1098: the sarcolemmal K_ATP channel blocker was administered 5 min before Postcon. After 24 h of R, infarct size was smaller in Postcon relative to Control (27 ± 4%* Vs. 39 ± 2% of area at risk), consistent with a reduction in CK activity (66 ± 7* Vs. 105 ± 7 IU/g). The infarct-sparing effect of Postcon was blocked by 5-HD (48 ± 5%^†), but was not altered by HMR1098 (29 ± 3%*), consistent with the change in CK activity (102 ± 8^† in 5-HD and 71 ± 6* IU/g in HMR1098). In H9c2 cells exposed to 8 h hypoxia and 3 h of reoxygenation, Postcon up-regulated expression of mito-K_ATP channel Kir6.1 protein, maintained mitochondrial membrane potential and inhibited mitochondrial permeability transition pore (mPTP) opening evidenced by preserved fluorescent TMRE and calcein staining. The protective effects were blocked by 5-HD, but not by HMR1098. These data suggest that in a clinically relevant model of ischemia-reperfusion (1) Postcon reduces infarct size and decreases CK activity after prolonged reperfusion; (2) protection by Postcon is achieved by opening mitochondrial K_ATP channels and inhibiting mPTP opening. *P < 0.05 Vs. Control; ^† P < 0.05 Vs. Postcon.