Cardiomyocyte apoptosis is a component of cardiac remodeling that can contribute to heart failure in obesity. A role for leptin in mediating this process has been suggested and the objective of this work was to investigate the effect of leptin on apoptosis and associated mechanisms in H9c2 cells which were subjected to hypoxia/reoxygenation (HR) to mimic myocardial ischemia/reperfusion. Qualitative immunofluorescent and quantitative laser scanning cytometry approaches demonstrated that exposure of cells to HR increased DNA fragmentation (TUNEL staining) which was attenuated by leptin (6 nM, 1 h) pretreatment. We also found increased annexin-V binding and caspase-3 activity in cells exposed to HR, both of which were attenuated by leptin pretreatment. Leptin reduced HR-induced translocation of the pro-apoptotic protein Bax to the mitochondrial membrane, which provides a mechanism to explain its protective effect. Consequently, leptin attenuated the HR-induced decrease in mitochondrial membrane potential and increase in cytochrome c release from mitochondria. Leptin treatment increased the phosphorylation of p38 MAPK and AMPK and respective inhibitors of these kinases, SB203580 and Compound C, prevented the ability of leptin to decrease HR-induced caspase-3 activity. In conclusion, we establish mechanisms via which leptin exerts anti-apoptotic effects that may be of significance in understanding the development of heart failure in obesity.