The Cdc25C phosphatase mediates cellular entry into mitosis in mammalian cells. Cdc25C activates Cdc2 for entry into mitosis by dephosphorylating Thr and Tyr at the site of inhibitory phosphorylation. The Cdc25C gene contains tumor suppressor p53 binding sites and is demonstrated to contribute to the p53-dependent cell cycle arrest upon DNA damage. Here we show that both Cdc25C and Cdc2 were down-regulated in wild-type HCT116 cells but not in p53-null, DNMT1-null or DNMT1and DNMT3b-null cells, upon p53 stabilization following doxorubicin-mediated DNA damage. Furthermore, zebularine, a drug that selectively traps and depletes nuclear DNMT1 and DNMT3b, relieved p53-mediated repression of endogenous Cdc25C and Cdc2. Methylation analysis of the Cdc25C and Cdc2 promoter displayed internal CG methylation proximal to the p53 binding site upon DNA damage in a p53-dependent manner. Chromatin immunoprecipitation of doxorubicin treated wild-type HCT116 cells showed the presence of DNMT1, p53, H3K9me2, and the transcriptional repressor HDAC1 on the Cdc25C and Cdc2 promoters, suggesting their involvement as repressive complexes in Cdc25C and Cdc2 gene silencing. Thus, the general mechanism of p53-mediated gene repression may involve recruitment of other repressive factors.