Melatonin has been shown to protect against ischemia/reperfusion (I/R)‐induced myocardial injury, however, the precise molecular mechanisms have not been fully clarified. The present study was aimed to investigate whether inactivation of Rac1/JNK/Foxo3a/Bim signaling pathway is responsible for the protective effect of melatonin on I/R‐induced myocardial injury. Our results showed that Foxo3a downregulation contributed to the protective effect of melatonin on OGD/R‐induced injury of H9c2 cardiomyoblasts. Melatonin treatment led to a reduced activity of Rac1, which was responsible for Foxo3a downregulation and decreased cell injury in OGD/R‐exposed H9c2 cells. Furthermore, JNK acts as a downstream effector of Rac1 in mediating melatonin‐induced inactivation of Foxo3a/Bim signaling pathway and decreased cell injury in OGD/R‐exposed H9c2 cells. In conclusion, our results indicate that melatonin protects H9c2 cells against OGD/R‐induced injury by inactivating the Rac1/JNK/Foxo3a/Bim signaling pathway. This study provided a novel insight into the protective mechanism of melatonin against I/R‐induced myocardial injury.