The ATP-sensitive K⁺ channels (K_ATP) in both sarcolemmal (sarcK_ATP) and mitochondrial inner membrane (mitoK_ATP) are the critical mediators in cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcK_ATP contains Kir6.2 and SUR2A, the molecular identity of mitoK_ATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing K_ATP into mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A showed that Kir6.2-containing K_ATP channels were localized in mitochondria, and this mitochondrial localization was significantly increased by PKC activation with PMA. FRET microscopy further revealed that significant amount of Kir6.2-containing K_ATP channels were localized in mitochondrial inner membrane after PKC activation. These results were supported by western blotting showing that Kir6.2 protein level in mitochondria from COS-7 cells transfected with Kir6.2/SUR2A was enhanced after PMA treatment, and this increase was inhibited by selective PKC inhibitor chelerythrine. Furthermore, functional analysis indicated that the number of functional K_ATP channels in mitochondria was significantly increased by PMA, as shown by K_ATP-dependent decrease in mitochondrial membrane potential in COS-7 cells transfected with Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in mitochondrial Kir6.2-containing K_ATP channels were blocked by a selective PKC inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the K_ATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria and the Kir6.2 content in mitochondria is increased by activation of PKC. PKC isoform -regulated mitochondrial import of K_ATP channels may have significant implication in cardioprotection of IPC.