The NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2α physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2α, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2α represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2α point mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a p53 regulatory pathway mediated by mammalian Sir2α. These results have significant implications regarding an important role for Sir2α in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.