ABSTRACT: The glucose transporter GLUT4 plays central roles in maintaining blood glucose homeostasis. Under basal conditions, GLUT4 is sequestered in intracellular vesicles in adipose and skeletal muscle cells. Upon insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 to the cell surface. Once on the cell surface, GLUT4 facilitates the uptake of excess blood glucose into the cell for disposal, thereby maintaining the blood glucose homeostasis. Imbalances in GLUT4 exocytosis result in insulin resistance and type 2 diabetes. GLUT4 vesicle fusion requires SNAREs as the core machinery, and a large group of regulatory factors. It remains unclear how SNAREs act in concert with the regulatory factors to mediate and regulate GLUT4 vesicle fusion. We are addressing the problem from a novel angle by reconstituting GLUT4 vesicle fusion in vitro using purified components, and then validating our findings in adipocytes. In defined fusion systems, SNAREs and regulatory factors can be individually added or perturbed without the complications of other molecules naturally present in the cell, allowing their kinetic effects on fusion to be causally established. We have reconstituted and characterized eight proteins implicated in GLUT4 exocytosis including three SNAREs – syntaxin-4, SNAP-23, VAMP2– and five regulatory factors – Doc2b, Munc18c, E-Syt1, tomosyn, and synip. We observed that GLUT4 exocytic SNAREs drive a basal membrane fusion reaction. Each of the regulatory factors binds to a distinct assembly of the SNAREs on the membrane and markedly alters fusion kinetics. Whereas Doc2b, Munc18c and E-Syt1 robustly accelerate the fusion kinetics, tomosyn and synip arrest membrane fusion at distinct intermediate stages. Our findings provide novel mechanistic insights into insulin-controlled GLUT4 exocytosis, and may facilitate the understanding and treatment of insulin resistance and type 2 diabetes.