Human cdc25C is a dual-specificity phosphatase involved in the regulation of cell cycle progression in both unperturbed cells and in cells subject to DNA damage or replication checkpoints. In this study, we describe the structure-function relationship of an essential domain of human cdc25C that interacts with 14-3-3 proteins. We show that this domain is a bi-functional interactive motif that interacts with cyclins primarily through their P-box motif in addition to 14-3-3 proteins. Characterization of the structural features of this domain by NMR and circular dichroism reveals two distinct alpha helical moieties interconnected by a loop carrying the 14-3-3 binding site. Moreover, the helical folding is induced upon binding to 14-3-3, suggestive of a conformational regulation of this domain of cdc25C through interactions with partner proteins in vivo. Combining our structural and biochemical data, we propose a detailed model of the molecular mechanism of cdc25C regulation by differential association with 14-3-3 and cdc2-cyclin B.