The regulated delivery of Glut4-containing vesicles to the plasma membrane is a specialised example of regulated membrane trafficking. Present models favour the transporter trafficking through two inter-related endosomal cycles. The first is the proto-typical endosomal system. This is a fast trafficking event that, in the absence of insulin, serves to internalise Glut4 from the plasma membrane. Once in this pathway, Glut4 is further sorted into a slowly recycling pathway that operates between recycling endosomes, the trans Golgi network, and a population of vesicles often referred to as Glut4-storage vesicles. Little is known about the molecules that regulate these distinct sorting steps. Here, we have studied the role of Stx16 in Glut4 trafficking. Using two independent strategies, we show that Stx16 plays a crucial role in Glut4 traffic in 3T3-L1 adipocytes. Over-expression of a mutant form of Stx16 devoid of a transmembrane anchor was found to significantly slow the reversal of insulin-stimulated glucose transport. Depletion of Stx16 using antisense approaches profoundly reduced insulin-stimulated glucose transport but was without effect on cell surface transferrin receptor levels, and also reduced the extent of Glut4 translocation to the plasma membrane in response to insulin. These data support a model in which Stx16 is crucial in the sorting of Glut4 from the fast cycling to the slow cycling intracellular trafficking pathways in adipocytes.