Esculetin (6,7-dihydroxycoumarin) is a derivative of coumarin compound, and has been shown to encompass a variety of pharmacological activities. Little is known about the cardio-protective action of esculetin as well as its molecular mechanisms. In this study, we investigated the effects of esculetin on cardiomyocyte cell apoptosis in response to hypoxia/reoxygenation (H/R) injury, and the potential molecular mechanisms were also explored. Our results showed that esculetin recovered cell viability in H9c2 cells after H/R injury. Furthermore, esculetin inhibited oxidant stress and cell apoptosis in H9c2 cells after H/R injury. Lastly, esculetin significantly reduced the phosphorylation levels of p38, ERK and JNK in H9c2 cells following H/R treatment. In conclusion, the present study demonstrated that esculetin attenuates myocardial ischemia/reperfusion (I/R) injury by inhibiting oxidative stress and cardiomyocyte apoptosis via downregulation of the MAPK signaling pathway. Thus, these findings suggest that esculetin may be an effective therapeutic agent for the prevention or treatment of myocardial I/R injury.