During differentiation, expression of protein phosphatase-2Cα (PP2Cα) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Cα in insulin signaling, we overexpressed wild-type (WT) PP2Cα by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Cα-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Cα-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Cα(R174G) did not produce such effects. Furthermore, overexpression of PP2Cα-WT (but not PP2Cα(R174G)) decreased the 32P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Cα activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Cα by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Cα antibody or PP2Cα small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Cα is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.