It is established that wortmannin which completely inhibits class IA PI 3-kinase activation abrogated the insulin-dependent translocation of GLUT4 to the plasma membrane in adipocytes and skeletal muscle. However, it was not clear which steps wortmannin inhibited during the whole translocation process of GLUT4. We have now dissected the each steps of the GLUT4 trafficking in 3T3-L1 adipocytes using exogenously-expressed GLUT4 reporter in combination with plasma membrane lawn assay. We showed that 100 nM wortmannin inhibited the fusion of GLUT4 vesicles to the plasma membrane without affecting the movement and the subsequent tethering/docking event of GLUT4 vesicles to the membrane in 3T3-L1 adipocytes. These results suggest that wortmannin-sensitive insulin signaling pathway plays a crucial role in the fusion step of GLUT4 vesicles to the plasma membrane in 3T3-L1 adipocytes.