Mitochondrial dysfunction caused by calcium overload is a vital factor for mediating cardiomyocyte death following ischemia/reperfusion (I/R) injury. The stromal interactive molecule 2 (STIM2) is a calcium sensor protein that regulates the store-operated calcium entry (SOCE). Whereas, whether STIM2 is associated with I/R injury remains largely unclear. We report here that STIM2, but not its homologue STIM1, is upregulated in cultured H9c2 cells, a cell model for cardiomyocytes, following I/R injury. In addition, the knockdown of STIM2, but not STIM1, reduces H9c2 cell apoptosis following I/R injury, and similar results were obtained in primary neonatal cardiomyocytes. This anti-apoptotic effect could be attributed to the inhibited activation of mitochondrial apoptosis pathway. Moreover, STIM2 knockdown reduces ER calcium release and simultaneously alleviates mitochondrial calcium overload in H9c2 cells following I/R injury. Furthermore, STIM2 knockdown decreases mitochondrial injury and preserves mitochondrial function following I/R injury. Collectively, these results suggest that the protective role of STIM2 knockdown against I/R injury in cardiomyocytes is associated with the reduced mitochondrial calcium overload and preserved mitochondrial function. Hence, our study may provide a novel insight into the regulation of mitochondrial-mediated cardiomyocyte apoptosis following I/R injury. Copyright © 2019. Published by Elsevier Inc.