Background/Aims: Nicotinamide plays a protective role in hypoxia-induced cardiomyocyte dysfunction. However, the underlying molecular mechanisms remain poorly understood. The purpose of this study was to investigate these and the effect of nicotinamide pretreatment on hypoxic cardiomyocytes. Methods: Cultured rat cardiomyocytes were pretreated with nicotinamide, subjected to hypoxia for 6 h, and then cell necrosis and apoptosis were examined. The effects of nicotinamide pretreatment on hypoxia-induced reactive oxygen species (ROS) formation, antioxidant enzyme expression, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) levels, adenosine triphosphate (ATP) production and mitochondrial membrane potential were tested to elucidate the underlying mechanisms. Results: Based on the findings that nicotinamide treatment decreased protein expression of receptor-interacting protein (RIP; a marker for cell necrosis) and cleaved caspase-3 (CC3; a marker for cell apoptosis) in normoxic cardiomyocytes, we found that it dramatically reduced hypoxia-induced necrosis and apoptosis in cardiomyocytes. The underlying mechanisms of these effects are associated with the fact that it increased protein expression of superoxide dismutase and catalase, increased intracellular levels of NAD+ and ATP concentration, decreased mitochondrial ROS generation and prevented the loss of mitochondrial membrane potential. Conclusion: All of these results indicate that nicotinamide pretreatment protects cardiomyocytes by improving mitochondrial stress. Our study provides a new clue for the utilization of nicotinamide in therapies for ischemic heart disease.