—Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoK_ATP channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoK_ATP channel diazoxide attenuates the lethal injury associated with Ca²⁺overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoK_ATP channels; whether diazoxide mimics the effects of Ca²⁺ preconditioning; and whether diazoxide reduces Ca²⁺ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca²⁺ PD (10 minutes of Ca²⁺ depletion followed by 10 minutes of Ca²⁺ repletion). The effects of the MitoK_ATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca²⁺ PD were assessed. In hearts treated with 80 μmol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca²⁺ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca²⁺ PD hearts. The salutary effects of diazoxide on the Ca²⁺ PD injury were similar to those in hearts that underwent Ca²⁺ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca²⁺ PD. The addition of sodium 5-hydroxydecanoate, a specific MitoK_ATP channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca²⁺ PD. Blockade of Ca²⁺ entry during diazoxide treatment by inhibiting L-type Ca²⁺ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca²⁺ PD. PKC-δ was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-α and PKC-ε were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoK_ATP channel is mediated by PKC-mediated signaling pathway.