Oxidative stress-mediated cell death in cardiomyocytes reportedly plays an important role in many cardiac pathologies. Our previous report demonstrated that mitochondrial SIRT3 plays an essential role in mediating cell survival in cardiac myocytes, and that resveratrol protects cardiomyocytes from oxidative stress-induced apoptosis by activating SIRT3. However, the exact mechanism by which SIRT3 prevents oxidative stress remains unknown. Here, we show that exposure of H9c2 cells to 50μM H₂O₂ for 6h caused a significant increase in cell death and the down-regulation of SIRT3. Reactive oxygen species (ROS)-mediated NF-κB activation was involved in this SIRT3 down-regulation. The SIRT3 activator, resveratrol, which is considered an important antioxidant, protected against H₂O₂-induced cell death, whereas the SIRT inhibitor, nicotinamide, enhanced cell death. Moreover, resveratrol negatively regulated H₂O₂-induced NF-κB activation, whereas nicotinamide enhanced H₂O₂-induced NF-κB activation. We also found that SOD2, Bcl-2 and Bax, the downstream genes of NF-κB, were involved in this pathological process. These results suggest that SIRT3 protects cardiomyocytes exposed to oxidative stress from apoptosis via a mechanism that may involve the NF-κB pathway.