Astrocytes are responsible for clearance of extracellular glutamate, primarily through glial-specific glutamate transporter-1 and the Na(+)-dependent glutamate/aspartate transporter (GLAST). After traumatic injury to the CNS, such as spinal cord injury, persistent release of ATP from damaged neurons and activated glial cells occurs, inducing detrimental and/or beneficial effects via activation of ionotropic (P2XR) and metabotropic purinergic receptors. In this study, we show a decrease in GLAST mRNA in the lesion center and caudal portions at 24 h post-spinal cord injury. In an in vitro system, the ability of astrocytes to take up glutamate and astrocytic GLAST mRNA levels were significantly decreased after exposure to ATP and its P2X(7)R agonist, 2'-3'-O-(4-benzoylbenzoyl)-ATP. ATP- or 2'-3'-O-(4-benzoylbenzoyl)-ATP-induced inhibitory effect on GLAST mRNA expression was blocked by the irreversible P2X(7)R blocker, oxidized ATP, or when P2X(7)R mRNA expression was reduced by the lentivirus-short hairpin RNA knockdown approach. Furthermore, deletion of the GLAST promoter and RNA decay assays showed that P2X(7)R signaling triggered post-transcriptional regulation of GLAST expression via the phosphoinositide 3-kinase cascade. The signaling pathway participating in the P2X(7)R effect on GLAST mRNA expression was identified as a Ca(2+)-dependent phosphoinositide 3-kinase-phospholipase Cgamma involving the inositol 1,4,5-trisphosphate receptor, calcium/calmodulin-dependent kinase II, and protein kinase C. We conclude that P2X(7)R activation by sustained release of ATP in the injured CNS may decrease GLAST mRNA stability via Ca(2+)-dependent signaling, suggesting that inhibition of P2X(7)R may allow for recovery of astrocytic GLAST function and protect neurons from glutamate-induced excitotoxicity.