Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation.