An orchestrated signaling cascade termed the DNA damage response (DDR) leads to the cellular response to damage, including cell cycle arrest, DNA repair and induction of apoptosis. Etoposide is widely used in the treatment of cancer including leukemia. Pluripotent stem cells have a highly efficient DNA repair system that becomes less efficient during differentiation. SIRT1, also known as NAD-dependent deacetylase, involved in various different normal physiologic and disease processes. In this study, we evaluated whether SIRT1 had an effect on embryonic stem cell programmed death in response to DNA damage induced by etoposide. Phosphorylation of H2AX, Chk1, and P53 was more strongly activated in Sirt1+/+ mESC than in Sirt1-/- mESC at equivalent doses of etoposide. Acetylation of P53 was more increased in Sirt1-/- mESC than that of Sirt1+/+ mESC. Decreae of SIRT1 reduced accumulation etoposide-induced intracellular reactive oxygen species. Moreover, flow cytometric analysis of cell cycle progression demonstrated that SIRT1 extend the cell cycle arrest induce by etoposide. The protein levels of LC3-II was down-regulated in Sirt1-/-mESC. Taken together, these findings indicate that SIRT1 modulates embryonic stem cell programmed cell death in response to DNA damage