Glutamate uptake by the Na+-dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial–neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1–EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N-methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC50 value of glutamate was 240 μm. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na+-free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L-trans-pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na+/K+-ATPase inhibitor, ouabain, and the Na+ ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca2+signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na+ influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes.