Ischemia/reperfusion injury (IRI) is harmful to the cardiovascular system and causes mitochondrial oxidative stress. Numerous data indicate that the JAK2/STAT3 signaling pathway is specifically involved in preventing myocardial IRI. Melatonin has potent activity against IRI and may regulate JAK2/STAT3 signaling. This study investigated the protective effect of melatonin pretreatment on myocardial IRI and elucidated its potential mechanism. Perfused isolated rat hearts and cultured neonatal rat cardiomyocytes were exposed to melatonin in the absence or presence of the JAK2/STAT3 inhibitor AG490 or JAK2 siRNA and then subjected to IR. Melatonin conferred a cardio-protective effect, as shown by improved postischemic cardiac function, decreased infarct size, reduced apoptotic index, diminished lactate dehydrogenase release, up-regulation of the anti-apoptotic protein Bcl2, and down-regulation of the pro-apoptotic protein Bax. AG490 or JAK2 siRNA blocked melatonin-mediated cardio-protection by inhibiting JAK2/STAT3 signaling. Melatonin exposure also resulted in a well-preserved mitochondrial redox potential, significantly elevated mitochondrial superoxide dismutase (SOD) activity, and decreased formation of mitochondrial hydrogen peroxide (H2O2) and malondialdehyde (MDA), which indicates that the IR-induced mitochondrial oxidative damage was significantly attenuated. However, this melatonin-induced effect on mitochondrial function was reversed by AG490 or JAK2 siRNA treatment. In summary, our results demonstrate that melatonin pretreatment can attenuate IRI by reducing IR-induced mitochondrial oxidative damage via the activation of the JAK2/STAT3 signaling pathway.