Insulin stimulates glucose uptake into muscle and fat cells by translocating glucose transporter 4 (GLUT4) to the cell surface, with input from phosphatidylinositol (PI) 3-kinase and its downstream effector Akt/protein kinase B. Whether PI 3,4,5-trisphosphate (PI(3,4,5)P3) suffices to produce GLUT4 translocation is unknown. We used two strategies to deliver PI(3,4,5)P3 intracellularly and two insulin-sensitive cell lines to examine Akt activation and GLUT4 translocation. In 3T3-L1 adipocytes, the acetoxymethyl ester of PI(3,4,5)P3 caused GLUT4 migration to the cell periphery and increased the amount of plasma membrane-associated phospho-Akt and GLUT4. Intracellular delivery of PI(3,4,5)P3 using polyamine carriers also induced translocation of myc-tagged GLUT4 to the surface of intact L6 myoblasts, demonstrating membrane insertion of the transporter. GLUT4 translocation caused by carrier-delivered PI(3,4,5)P3 was not reproduced by carrier-PI 4,5-bisphosphate or carrier alone. Like insulin, carrier-mediated delivery of PI(3,4,5)P3 elicited redistribution of perinuclear GLUT4 and Akt phosphorylation at the cell periphery. In contrast to its effect on GLUT4 mobilization, delivered PI(3,4,5)P3 did not increase 2-deoxyglucose uptake in either L6GLUT4myc myoblasts or 3T3-L1 adipocytes. The ability of esogenously delivered PI(3,4,5)P3 to augment plasma membrane GLUT4 content without increasing glucose uptake suggests that input at the level of PI 3-kinase suffices for GLUT4 translocation but is insufficient to stimulate glucose transport.