Myocardial ischemia reperfusion injury (I/RI) can lead to acute myocardial infarction (MI), which is a common cardiovascular disease (CVD) leading to morbidity and mortality worldwide. Betulinic acid exerts protective effect on myocardial I/RI in rats; however, the mechanism remains unknown. The aim of the current study was to evaluate the effect of betulinic acid on myocardial I/RI in vitro and further investigate the underlying mechanism. Our results showed that betulinic acid increased cell viability and decreased lactate dehydrogenase (LDH) release in H9c2 cells exposed to H/R. Betulinic acid was found to suppress the production of oxidative stress markers in H/R-induced H9c2 cells. The H/R-mediated changes in caspase-3 activity and expressions of Bax and Bcl-2 were restored by betulinic acid treatment. Besides, betulinic acid enhanced the activation of Nrf2/HO-1 pathway, and inhibited the activation of p38 and JNK pathways in H/R-induced H9c2 cells. Moreover, treatment with Nrf2 specific siRNA or the specific inhibitors of p38 and JNK attenuated the protective effects of betulinic acid on H9c2 cells. The results denoted that betulinic acid protects the H9c2 cells from I/RI by inhibiting oxidative stress and cell apoptosis. The cardio-protective effects were mediated by the Nrf2/HO-1, p38 and JNK pathways.