Clinical insulin resistance is associated with decreased activation of phosphatidylinositol 3'-kinase (PI3K) and its downstream substrate protein kinase B (PKB)/Akt. However, its physiological protein substrates remain poorly characterized. In the present study, the effect of in vivo insulin action on phosphorylation of the PKB/Akt substrate 40 (PRAS40) was examined. In rat and mice, insulin stimulated PRAS40-Thr246 phosphorylation in skeletal and cardiac muscle, the liver, and adipose tissue in vivo. Physiological hyperinsulinemia increased PRAS40-Thr246 phosphorylation in human skeletal muscle biopsies. In cultured cell lines, insulin-mediated PRAS40 phosphorylation was prevented by the PI3K inhibitors wortmannin and LY294002. Immunohistochemical and immunofluorescence studies showed that phosphorylated PRAS40 is predominantly localized to the nucleus. Finally, in rats fed a high-fat diet (HFD), phosphorylation of PRAS40 was markedly reduced compared with low-fat diet-fed animals in all tissues examined. In conclusion, the current study identifies PRAS40 as a physiological target of in vivo insulin action. Phosphorylation of PRAS40 is increased by insulin in human, rat, and mouse insulin target tissues. In rats, this response is reduced under conditions of HFD-induced insulin resistance.