Previous studies have shown that stromal interaction molecule1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation in H9C2 cells subjected to hypoxia/reoxygenation(H/R) injury. The aim of the present study was to investigate the effect of resveratrol on STIM1-mediated intracellular Ca2+ accumulation and subsequent cell death in the context of myocardial ischemia/reperfusion (I/R) injury. C57 BL/6 mice were fed with either saline or resveratrol (50 mg/kg daily for 2 weeks) and then subjected to myocardial I/R injury. TTC/Evans Blue staining and TUNEL assay were performed to quantify the infarct size and apoptosis index. The cardiac function was evaluated by echocardiography. Neonatal rat ventricular cardiomyocytes (NRVCs) underwent hypoxia/reoxygenation (H/R) to establish the in vitro model. To achieve over-expression, NRVCs were transfected with STIM1-adenovirus vector. Apoptosis was analyzed by TUNEL assay. Cell viability was measured using MTS assay and cell necrosis was determined by LDH release assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using a Fluo-3AM probe. Resveratrol significantly reduced apoptosis, decreased infarct size, and improved cardiac function in mice subjected to myocardial I/R injury. In NRVCs, resveratrol also downregulated STIM1 expression accompanied by decreased intracellular Ca2+ accumulation elicited by H/R injury. In addition, resveratrol reduced cell apoptosis, upregulated the Bcl-2, decreased Bax, and cleaved caspase-3 expression. Furthermore, the effects of resveratrol on STIM1-mediated intracellular Ca2+ accumulation, apoptotic proteins, and H/R-induced cell injury were exacerbated by STIM1 over-expression and were partly abolished by SOCE inhibitor SKF96365 in NRVCs in vitro. Our findings demonstrate that resveratrol exerts anti-apoptotic activity and improves cardiac functional recovery following myocardial I/R by inhibiting STIM1-induced intracellular Ca2+ accumulation.