From time of their discovery, sarcolemmal K_ATP (sarcK_ATP) channels were thought to have an important protective role in the heart during stress whereby the channel opening protects the heart from stress-induced Ca²⁺ overload and resulting damage. In contrast, some of the recent studies indicate that the sarcK_ATP channel closing can lead to cardiac protection. Also, the role of the sarcK_ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK_ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK_ATP channel inhibitor HMR-1098 as evidenced by TUNEL and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol suggesting activation of the mitochondrial death pathway. SarcK_ATP channel inhibition during stress had no effect on Bcl-2, Bad and p-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of apoptotic pathway. However, monitoring of mitochondrial Ca²⁺ using rhod-2 fluorescent indicator revealed that mitochondrial Ca²⁺ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK_ATP channels, through a specific Ca²⁺-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.