P53 upregulated modulator of apoptosis (PUMA) plays an important role in mediating cell death. However, the role of PUMA in cardiomyocyte death induced by hypoxia/reoxygenation (H/R) and its molecular mechanism still remain enigmatic. Here, we used the in vitro model to elucidate the effects of PUMA on H/R-induced cardiomyocyte apoptosis as well as the underlying mechanisms. We reported that H/R could upregulate the expression of PUMA accompanied by the elevation of cardiomyocyte apoptosis. Interestingly, inhibition of endogenous PUMA expression by PUMA siRNA or p53 inhibitor repressed H/R-induced cardiomyocyte apoptosis. Furthermore, we found H/R stimulated the associations of PUMA apoptosis repressor with caspase recruitment domain (ARC) and consequently attenuated the associations of ARC with caspase 8, resulting in caspase 8 activation. Also, H/R stimulated cytochrome C release and caspase 3 activation. However, these stimulating effects of H/R disappeared upon knockdown of endogenous PUMA. Our data reveal that PUMA participates in H/R-triggered cardiomyocyte apoptosis by interfering with mitochondrial pathway.