ATP has been recognized as an important extracellular signaling molecule and P2X receptors are membrane ion channels activated by the binding of extracellular ATP. Since both AQP4 and P2X7 receptor (P2X7R) are known to be present in astrocytes, we examined whether P2X7R activation plays a role in the regulation of AQP4 expression in astrocytes. Immunoblotting and immunocytochemistry confirmed the expression of both P2X7R and AQP4 in primary cultured rat astrocytes. Co-immunoprecipitation assays of the HEK293 cells expressing both proteins revealed no protein-protein interaction. An activation of P2X7R in primary cultured astrocytes by a P2X7R agonist significantly decreased the AQP4 protein expression, which was abolished by the pre-treatment of a P2X7R antagonist. In addition, AQP4 expression was not affected by high extracellular copper, zinc, or iron concentrations. In a rat model with anoxia-induced brain injury where extracellular ATP levels could be increased, whole brain AQP4 expression was significantly decreased, whereas P2X7R expression was unchanged. Importantly, pre-treatment of P2X7R antagonist in rats significantly inhibited the AQP4 down-regulation in anoxic brain injury, consistent with the in vitro results observed in astrocytes. In conclusion, P2X7R activation in astrocytes was associated with down-regulation of AQP4 in rat brain astrocytes in vitro and in vivo, and this was prevented by P2X7 receptor blockade. Thus, an activation of P2X7R in astrocytes in response to brain injury is likely to play a role in the protective down-regulation of AQP4, which might inhibit water influx to the cells and attenuate the acute cytotoxic brain edema after acute brain injury.