Growing evidence indicates that metabolic signaling pathways are interconnected to DNA damage response (DDR). However, factors that link metabolism to DDR remain incompletely understood. SIRT1, an NAD+-dependent deacetylase that regulates metabolism and aging, has been shown to protect cells from DDR. Here, we demonstrate that SIRT1 protects cells from oxidative stress-dependent DDR by binding and deacetylating checkpoint kinase 2 (CHK2). We first showed that essential proteins in DDR were hyperacetylated in Sirt1-deficient cells and that among them, the level of acetylated CHK2 was highly increased. We found that Sirt1 formed molecular complexes with CHK2, BRCA1/BRCA2-associated helicase 1 (BACH1), tumor suppressor p53-binding protein 1 (53BP1), and H2AX, all of which are key factors in response to DNA damage. We then demonstrated that CHK2 was normally inhibited by SIRT1 via deacetylation but dissociated with SIRT1 under oxidative stress conditions. This led to acetylation and activation of CHK2, which increased cell death under oxidative stress conditions. Our data also indicated that SIRT1 deacetylated the K235 and K249 residues of CHK2, whose acetylation increased cell death in response to oxidative stress. Thus, SIRT1, a metabolic sensor, protects cells from oxidative stress-dependent DDR by the deacetylation of CHK2. Our findings suggest a crucial function of SIRT1 in inhibiting CHK2 as a potential therapeutic target for cancer treatment.