Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G(2) checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.