Speedy (Spy1) is a novel cell cycle regulator that binds and activates cdk2, and was originally identified as a suppressor of Rad1 deficiency in Schizosaccharomyces pombe. Here we demonstrate that overexpression of human Spy1 enhances mammalian cell viability during cellular responses to DNA damage induced by genotoxic agents such as camptothecin, cisplatin, and hydroxyurea. Clonogenic survival assays and comet assays also show that Spy1 expression enhances cell survival after DNA damage. Consistent with Spy1 having a role in the DNA damage response, endogenous Spy1 protein levels are up-regulated in response to DNA damage induced by camptothecin, cisplatin, or hydroxyurea. We found that Spy1 can activate cdk2 during the DNA damage response and that expression of a dominant-negative form of cdk2 overrides Spy1 function in damaged cells. Lastly, ablation of endogenous Spy1 expression using small interference RNA results in hypersensitization of cells to DNA damage. Together, these results demonstrate that human Spy1 mediates protection of mammalian cells against DNA damage.