Cardiac ischemia/reperfusion injury is associated with reduced mitochondrial turnover and regeneration. There is currently no effective approach to stimulate mitochondrial biogenesis in the reperfused myocardium. In this study, we investigated whether melatonin could increase mitochondrial biogenesis and thus promote mitochondrial homeostasis in cardiomyocytes. Cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) injury with or without melatonin treatment, and various mitochondrial functions were measured. H/R injury repressed mitochondrial biogenesis in cardiomyocytes, whereas melatonin treatment restored mitochondrial biogenesis through the 5’ adenosine monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC1α) pathway. Melatonin enhanced mitochondrial metabolism, inhibited mitochondrial oxidative stress, induced mitochondrial fusion and prevented mitochondrial apoptosis in cardiomyocytes subjected to H/R injury. The melatonin-induced improvement in mitochondrial biogenesis was associated with increased cardiomyocyte survival during H/R injury. On the other hand, silencing of PGC1α attenuated the protective effects of melatonin on cardiomyocyte viability, thereby impairing mitochondrial bioenergetics, disrupting the mitochondrial morphology, and activating mitochondrial apoptosis. Thus, H/R injury suppressed mitochondrial biogenesis, while melatonin activated the AMPK/PGC1α pathway and restored mitochondrial biogenesis, ultimately protecting the reperfused heart.