Mdm2 oncoprotein plays a major role in inhibiting the p53 tumor suppressor protein. Here, we investigate phosphorylation of Mdm2 at serine 407 (S407). S407 is phosphorylated in cells after treatment with camptothecin (CPT) or hydroxyurea, inhibitors of DNA replication. S407 phosphorylation after CPT treatment is induced upon cell cycle arrest during S phase and prevented if entry into S phase of cell cycle is blocked. We found that a major kinase responsible for S407 phosphorylation is ATR, a DNA damage checkpoint protein that induces cell cycle arrest and promotes DNA repair in response to impaired DNA replication; induction of S407 phosphorylation is enhanced after expression of wild-type ATR, while it is inhibited by a dominant-negative form of ATR. Further, S407 is specifically phosphorylated by ATR in vitro. Substitution of S407 with aspartate (S407D), but not with alanine (S407A), promotes nuclear localization of p53. Taken together, our data indicate that S407 phosphorylation of Mdm2 by ATR reduces Mdm2-dependent export of p53 from nuclei to cytoplasm.