Inhibiting P2X4 receptor increases blood-brain barrier permeability and attenuates ischemic stroke. P2X4 receptor is involved in the modulation of blood-brain barrier (BBB) permeability. However, its roles in ischemic stroke are largely unknown. In this study, the levels of P2X4 receptor in mouse brain tissue and the effect of P2X4 receptor inhibitor on blood-brain barrier (BBB) permeability during cerebral ischemia were investigated. We found that P2X4 receptor was widely expressed in the mouse brain tissue and the expression of P2X4 receptor increased after ischemia and reperfusion. Intracerebroventricular injection of P2X4 receptor inhibitor BBG (1 mM) increased BBB permeability in sham group, especially in the cortex, hippocampus and thalamus. Brain water content, water content of the brain tissue and EB extravasation were all decreased after BBG administration. The infarct volumes were also reduced by BBG administration. The expression of P-selectin and aquaporin 4 (AQP4) decreased and Na+K+-ATPase increased in cerebral ischemic regions after BBG administration. The changes in P2X4 receptor expression and corresponding alterations of Na+K+-ATPase, P-selectin and AQP4 were all reversed by the co-administration of 5-BDBD, a selective antagonist of P2X4 receptor. These findings indicated that P2X4 receptor inhibitor BBG attenuated BBB permeability and reduced brain damage after cerebral ischemia/reperfusion. The mechanism underlying the regulation of P2X4 receptor on BBB permeability after cerebral ischemia/reperfusion was associated with the upregulation of Na+K+-ATPase and downregulation of P-selectin and AQP4.