Uncoupling protein-2 (UCP2) is an inner-mitochondrial membrane proton carrier that uncouples ATP synthesis. The aim of this study was to determine if UCP2 plays a role in survival of adult rat cardiac myocytes. We first studied the effects of UCP2 overexpression in vitro. Overexpression of UCP2 in primary cardiomyocytes leads to a significant decline in ATP level and the development of acidosis, but had no observable effect on cell survival. When cardiomyocytes were challenged with hypoxia/reoxygenation, cells overexpressing UCP2 survived significantly less compared to the control. This finding was associated with upregulation of pro-apoptotic protein BNIP3. Furthermore, UCP2 siRNA prevented both the increase in cell death and BNIP3 expression. To examine the in vivo role of UCP2 in the heart, we used the Dahl salt-sensitive (DSS) rat heart failure model. Northern blot analysis revealed that UCP2 mRNA level was significantly upregulated in rat heart failure along with BNIP3 protein level. In conclusion, UCP2 increases sensitivity of adult rat cardiac myocytes to hypoxia/reoxygenation, by way of ATP depletion and acidosis, which in turn causes accumulation of prodeath protein BNIP3.