G₁/S and G₂/M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G₂/M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G₂/M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G₂/M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li–Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G₂/M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G₂/M checkpoint is at least in part through modulation of the activity of the G₂-specific kinase, cyclin B1/p34^cdc2. Genetic and physiological evidence of a Gadd45-mediated G₂/M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G₂/M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G₂ arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 −/−) also retained a G₂/M checkpoint initiated by ionizing radiation and failed to arrest at G₂/M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G₂/M checkpoints in response to specific types of DNA damage.