Glucose transport is a highly regulated process and is dependent on a variety of signaling events. Glycogen synthase kinase-3 (GSK-3) has been implicated in various aspects of the regulation of glucose transport, but the mechanisms remain unclear. We have found that inhibition of basal GSK-3 activity (8-24 hr) in several cell types, including vascular smooth muscle cells, resulted in an approximately 2-fold increase in glucose uptake due to a similar increase in GLUT1 protein expression. Conversely, expression of a constitutively active form of GSK-3 led to a 2-fold decrease in GLUT1 expression and glucose uptake. Since GSK-3 can inhibit mTOR signaling via phosphorylation of the TSC2 tumor suppressor, we investigated whether chronic GSK-3 effects on glucose uptake and GLUT1 expression depended on phosphorylation of TSC2 and inhibition of mTOR. We found that absence of functional TSC2 resulted in a 1.5-3-fold increase in glucose uptake and GLUT1 expression. These increases in glucose uptake and GLUT1 levels were prevented by inhibition of mTOR with rapamycin. GSK-3 inhibition had no effect on glucose uptake or GLUT1 expression in TSC2 mutant cells, indicating that GSK-3 effects on GLUT1 and glucose uptake were mediated by a TSC2/mTOR-dependent pathway. The effect of GSK-3 inhibition on GLUT1 expression and glucose uptake was restored in TSC2 mutant cells by transfection of a wild-type TSC2 vector, but not by a TSC2 construct with mutated GSK-3 phosphorylation sites. Thus, TSC2 and rapamycin-sensitive mTOR function downstream of GSK-3 to modulate effects of GSK-3 on glucose uptake and GLUT1 expression.