The glutamate transporters GLAST and GLT-1 are mainly expressed in glial cells and regulate glutamate levels in the synapses. GLAST and GLT-1 are the targets of several signaling pathways. In this study we explore the possible functional interaction between these transporters and GSK3β. This kinase is involved in multiple cellular processes including neuronal development and synaptic plasticity. To evaluate whether GLT-1 and GLAST were regulated by GSK3β, we coexpressed these proteins in heterologous expression systems. In both COS-7 cells and Xenopus laevis oocytes, GSK3β stimulated the activity of GLT-1 and reduced that of GLAST. These effects were associated with corresponding changes in the amounts of GLT-1 or GLAST in the plasma membrane. These effects were suppressed by inhibitors of GSK3β or a catalytically inactive form of the kinase. GSK3β also decreases the incorporation of (32)Pi into GLT-1 and increases GLAST phosphorylation. Pharmacological inhibition of endogenous GSK3β in primary cultures of rat brain cortex also leads to a differential modulation of GLT-1 and GLAST. Our results suggest that constitutively active GSK3β is important in controlling the expression of functional glutamate transporters on the plasma membrane. This regulation might be relevant in physiological and pathological conditions in which glutamate transporters and GSK3β signaling are involved.