Sirtuin 6 (SIRT6) is a mammalian homolog of the yeast Sir2 deacetylase. Mice deficient for SIRT6 exhibit genome instability. Here, we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and stimulates DSB repair, through both nonhomologous end joining and homologous recombination. Our results indicate that SIRT6 physically associates with poly[adenosine diphosphate (ADP)–ribose] polymerase 1 (PARP1) and mono-ADP-ribosylates PARP1 on lysine residue 521, thereby stimulating PARP1 poly-ADP-ribosylase activity and enhancing DSB repair under oxidative stress.