The human Cdc25A phosphatase plays a pivotal role at the G1/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions.