Cell division cycle 25 (CDC25) phosphatases are key actors in eukaryotic cell cycle control. They are responsible for the dephosphorylations that activate the cyclin-dependent kinases (CDK) at specific stages of the cell cycle. Human CDC25A, CDC25B and CDC25C are also central targets and regulators of the G2/M checkpoint mechanisms activated in response to DNA injury. The expression and activity of these enzymes is finely regulated by multiple mechanisms including post-translational modifications, interactions with regulatory partners, control of their intracellular localization, and cell cycle-regulated degradation. Altered expression of these phosphatases is associated with checkpoint bypass and genetic instability. Accordingly, increased expression of CDC25A and CDC25B is found in many high-grade tumors and is correlated with poor prognosis in human cancers. This review summarizes our current knowledge within this domain and discusses the data that support therapeutic strategies targeting CDC25 activity in the treatment of cancer.