Astrocytes maintain glutamate homeostasis in the central nervous system (CNS) via glutamate uptake through Na(+)-dependent excitatory amino acid transporters (EAAT1 and EAAT2), and this process is regulated by several CNS cell types. However, it is unclear whether brain pericytes regulate glutamate uptake by astrocytes. Therefore, in this study, we aimed to investigate the effects of pericytes on the uptake of extracellular glutamate by astrocytes using an in vitro co-culture model of human brain-derived pericytes and astrocytes (pericyte co-cultures). The [(3)H]-L-glutamate ([(3)H]-L-Glu) uptake rate of astrocytes in pericyte co-cultures was significantly higher than that in astrocyte monocultures. Under Na(+)-free conditions, [(3)H]-L-Glu uptake by astrocytes was significantly inhibited in astrocyte monocultures and pericyte co-cultures. The inhibitory effect of Na(+) depletion on glutamate uptake by astrocytes was more pronounced in pericyte co-cultures than in astrocyte monocultures. These findings suggest that glutamate uptake by astrocytes through the Na(+)-dependent transporter EAATs is upregulated by pericytes. Treatment with dihydrokainic acid, a selective inhibitor of EAAT2, significantly inhibited [(3)H]-L-Glu uptake by astrocytes in pericyte co-cultures but not in astrocyte monocultures. Treatment with UCPH-101, a selective inhibitor of EAAT1, significantly inhibited [(3)H]-L-Glu uptake by astrocytes in both monocultures and pericyte co-cultures. The UCPH-101-induced reduction in [(3)H]-L-Glu uptake by astrocytes in pericyte co-cultures was similar to that observed in astrocyte monocultures. These results suggest that pericytes upregulate glutamate uptake via EAAT2 in astrocytes. Furthermore, [(3)H]-L-Glu uptake in astrocytes significantly increased when astrocytes were treated with pericyte-conditioned medium. This finding suggests that pericyte-derived soluble factors contribute to the upregulation of astrocytic glutamate uptake. To our knowledge, this is the first study to report that pericyte-released mediators upregulate the EAAT2-dependent uptake of extracellular glutamate in astrocytes.