Overexpression of Cdc25A phosphatase is often observed in cancer and results in poor prognosis. Cdc25A mainly dephosphorylates and thereby activates Cyclin-dependent kinase 2 and thus induces progression in the cell cycle from G(1) to S phase. Here, we demonstrate that the tumor suppressor p53 downregulates expression from the Cdc25A gene. In a p53-inducible cell system, Cdc25A expression on the mRNA and protein level is downregulated upon p53 expression. Promoter-reporter assays show that this regulation is dependent on the Cdc25A promoter. Mutant p53 fails to reduce Cdc25A transcription. In contrast to p53, neither p63 nor p73 can repress Cdc25A transcription. The Cdc25A promoter displays no p53 binding site, and p53 does not bind directly to the promoter DNA as shown by chromatin immunoprecipitation assays. Previously, the contribution of p53 to G(1)/S arrest has been mostly linked to activating the expression of the Cdk inhibitor p21(WAF1/CIP1). By downregulating Cdc25A expression, p53 may impair transition from G(1) to S phase independently of p21(WAF1/CIP1). Therefore, the data suggest that, as long as p53 is intact, Cdc25A transcriptional downregulation might play a role in cancer prevention.