Whether the mitochondrial ATP-dependent potassium (mK_ATP) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mK_ATP channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 µM), a selective mK_ATP channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 ± 3 vs. 35 ± 3% in control), indicating a role of mK_ATP channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca²⁺ channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 µM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 µM). The protection of diazoxide was not blocked by 50 µM 5-HD but was blocked by 200 µM 5-HD or 10 µM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mK_ATP opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mK_ATP channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca²⁺ transient but had no effect on L-type Ca²⁺ currents. Our results suggest that opening of mK_ATP channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca²⁺ and activation of PKC. During the lethal ischemia, mK_ATP channel opening mediates the protection, independent of PKC, by yet unknown mechanisms.