In Caenorhabditis elegans, fem-1, fem-2, and fem-3 play pivotal roles in sex determination. Recently, a mammalian homologue of the C. elegans sex-determining protein FEM-1, F1Aalpha, has been described. Although there is little evidence to link F1Aalpha to sex determination, F1Aalpha and FEM-1 both promote apoptosis in mammalian cells. Here we report the identification and characterization of a human homologue of the C. elegans sex-determining protein FEM-2, hFEM-2. Similar to FEM-2, hFEM-2 exhibited PP2C phosphatase activity and associated with FEM-3. hFEM-2 shows striking similarity (79% amino acid identity) to rat Ca(2+)/calmodulin (CaM)-dependent protein kinase phosphatase (rCaMKPase). hFEM-2 and FEM-2, but not PP2Calpha, were demonstrated to dephosphorylate CaM kinase II efficiently in vitro, suggesting that hFEM-2 and FEM-2 are specific phosphatases for CaM kinase. Furthermore, hFEM-2 and FEM-2 associated with F1Aalpha and FEM-1 respectively. Overexpression of hFEM-2, FEM-2, or rCaMKPase all mediated apoptosis in mammalian cells. The catalytically active, but not the inactive, forms of hFEM-2 induced caspase-dependent apoptosis, which was blocked by Bcl-XL or a dominant negative mutant of caspase-9. Taken together, our data suggest that hFEM-2 and rCaMKPase are mammalian homologues of FEM-2 and they are evolutionarily conserved CaM kinase phosphatases that may have a role in apoptosis signaling.