Newly synthesized GLUT4 enters into the insulin-responsive storage compartment in a process that is GGA (Golgi-localized -ear-containing Arf-binding protein)-dependent, whereas insulin-stimulated translocation is regulated by AS160 (Akt Substrate of 160 kDa). In the present study, using a variety of GLUT4/GLUT1 chimeras, we have analyzed the specific motifs of GLUT4 that are important for GGA and AS160 regulation of GLUT4 trafficking. Substitution of the amino terminal and the large intracellular loop of GLUT4 into GLUT1 (chimera 1–441) fully recapitulated the basal state retention, insulin-stimulated translocation and GGA and AS160 sensitivity of wild type GLUT4 (GLUT4-WT). GLUT4 point mutation (GLUT4-F5A) resulted in loss of GLUT4 intracellular retention in the basal state when co-expressed with both wild type GGA and AS160. Nevertheless, similar to GLUT4-WT, the insulin-stimulated plasma membrane localization of GLUT4-F5A was significantly inhibited by co-expression of dominant-interfering GGA. In addition, co-expression with a dominant-interfering AS160 (AS160–4P) abolished insulin-stimulated GLUT4-WT, but not GLUT4-F5A translocation. GLUT4 endocytosis and intracellular sequestration also required both the amino terminal and large cytoplasmic loop of GLUT4. Furthermore, both the FQQI and the SLL motifs participate in the initial endocytosis from the plasma membrane, however, once internalized, unlike the FQQI motif, the SLL motif is not responsible for intracellular recycling of GLUT4 back to the specialized compartment. Together, we have demonstrated that the FQQI motif within the amino terminus of GLUT4 is essential for GLUT4 endocytosis and AS160-dependent intracellular retention, but not for the GGA-dependent sorting of GLUT4 into the insulin-responsive storage compartment.