The cellular level of the CDC25A phosphatase is tightly regulated during both the normal and genotoxic-perturbed cell cycle. Here, we describe a caspase-dependent cleavage of this protein at residue D223 in non-genotoxic apoptotic conditions. This specific proteolysis generates a catalytically active C-terminal fragment that localizes to the nuclear compartment. Accumulation of this active CDC25A fragment leads to reduced inhibitory phosphorylation of the CDC25A substrate cyclin-dependent kinase 2 (CDK2) on Tyr15. Moreover, CDK2 was found stably associated with this fragment, as well as with an ectopically expressed CDC25A224-525 truncation mutant that mimicks the cleavage product. Ectopic expression of this mutant induced CDK2 Tyr15 dephosphorylation, whereas its catalytically inactive version did not. Finally, this 224-525 mutant initiated apoptosis when transfected into HeLa cells, whereas its catalytic inactive form did not. Altogether, this study demonstrates for the first time that caspase-dependent cleavage of CDC25A is a central step linking CDK2 activation with non-genotoxic apoptotic induction. The dual-specificity phosphatase CDC25A is an effector of key molecular steps during G1/S and M phases of the cell cycle. The initial function described for CDC25A was to remove the phosphate groups from the Thr14 and Tyr15 residues of cyclin-dependent kinase 2 (CDK2), allowing complete activation of this kinase and further progression of cells to S phase. 1,2 A similar function of CDC25A was also described during mitosis, participating in the activation of the Cdc2 kinase. 3 CDC25A is one of the three members of the CDC25 phosphatase family, which all directly activate CDKs throughout the cell cycle (for a recent review, see Boutros et al. 4 ). The specificity of CDC25A, over the B and C isoforms, is its essential function as a G1 phase regulator, and therefore, its regulation by extracellular signaling events. In addition, regulation through ubiquitin-dependent proteasomal degradation of the protein has been extensively described during the last few years, and participates in the G1/S cell cycle arrest and checkpoint (for a review, see Busino et al. 5 ). The cellular level of CDC25A is also dependent on mitogenic extracellular signals such as growth factors 6 and IL-7. 7 We also recently described that adhesion of acute myeloid leukemia (AML) cells to an extracellular matrix upregulates the level of this protein, leading to increased proliferation. 8 Less characterized than its role during the cell cycle are the potential functions of CDC25A during the apoptotic process. A pro-apoptotic role has been attributed to this phosphatase downstream of Myc-induced apoptosis, 9 with CDC25A being a direct transcriptional target of Myc in this context. Conversely, an antiapoptotic function has also been ascribed to the protein in response to serum starvation-induced cell death. 10 Interestingly, a direct interaction of CDC25A with the apoptosis signal regulated kinase-1 (ASK-1) was reported, leading to the inhibition ...