Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to synaptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular glutamate. We now report that adenosine A2A receptors (A2AR) control the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalemmal vesicles (gliosomes) from adult rats, whereas A1R and A3R were devoid of effects. Thus, the acute exposure to the A2AR agonist, CGS 21680, inhibited glutamate uptake, an effect prevented by the A2AR antagonist, SCH 58261, and abbrogated in cultured astrocytes from A2AR knockout mice. Furthermore, the prolonged activation of A2AR lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and protein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate transporters by astrocytic A2AR provides a novel candidate mechanism to understand the ability of A2AR to control synaptic plasticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters. © Wiley Periodicals, Inc.