Several studies have recently demonstrated that G protein-coupled estrogen receptor (GPER) 30 directly binds to estrogen and mediates its action. The aim of the present study was to investigate the effects of GPER on cardiocyte apoptosis following ischemia/reperfusion injury (MIRI) in H9C2 myocardial cells. H9C2 cells were treated with a specific GPER agonist (G1), 17 beta-estradiol (E2) or the vehicle. The cells were subjected to 20 min of myocardial ischemia followed by 120 min of reperfusion. They were then randomly assigned to three experimental groups: Control, G1, E2. B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X (Bax) levels were measured, Hoechst 33258 staining was performed to assess apoptosis, and superoxide dismutase (SOD), tumor necrosis factor (TNF)-alpha and adenosine triphosphatase (ATPase) levels were determined. To test the specificity of G1, GPER-knockout cells were treated with G1 and analyzed as stated above. Compared with the vehicle-treated groups, G1 and E2-treated groups exhibited elevated Bcl-2 levels, decreased Bax levels and cell apoptosis, significantly increased SOD and ATP levels and decreased TNF-alpha levels following ischemia-reperfusion. However, G1 had no evident effects on the GPER-knockout cells. In conclusion, the present study suggested that GPER activation provided a cardioprotective effect following ischemia-reperfusion by inhibiting cardiocyte apoptosis.