Mitochondrial ATP-sensitive K (mitoK_ATP) channels play a central role in protecting the heart from injury in ischemic preconditioning. In isolated mitochondria exposed to elevated extramitochondrial Ca, P_i, and anoxia to simulate ischemic conditions, the selective mitoK_ATP channel agonist diazoxide (25–50 μM) potently reduced mitochondrial injury by preventing both the mitochondrial permeability transition (MPT) and cytochrome c loss from the intermembrane space. Both effects were blocked completely by the selective mitoK_ATP antagonist 5-hydroxydecanoate. The protective effect against Ca-induced MPT was most evident under conditions in which the ability of electron transport to support membrane potential (Δψ_m) was decreased and inner membrane leakiness was increased moderately. Under these conditions, mitoK_ATP channel activity strongly regulated Δψ_m, and diazoxide prevented MPT by inhibiting the driving force for Ca uptake. Phorbol 12-myristate 13-acetate mimicked the protective effects of diazoxide, unless 5-hydroxydecanoate was present, indicating that protein kinase C activation also protects mitochondria by activating mitoK_ATP channels. Because Δψ_m recovery ultimately is required for heart functional recovery, these results may explain how mitoK_ATP channel activation mimics ischemic preconditioning by protecting mitochondria as they pass through a critical vulnerability window during ischemia/reperfusion.