We tested the hypothesis that the P2X4 purinergic receptor (P2X4) exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation after ischemia and reperfusion (IR). Supporting this, P2X4-deficient (KO) mice were protected against ischemic AKI with significantly attenuated renal tubular necrosis, inflammation, and apoptosis when compared to P2X4 wild-type (WT) mice subjected to renal IR. Furthermore, WT mice treated with P2X4 allosteric agonist ivermectin had exacerbated renal IR injury whereas P2X4 WT mice treated with a selective P2X4 antagonist (5-BDBD) were protected against ischemic AKI. Mechanistically, induction of kidney NLRP3 inflammasome signaling after renal IR was significantly attenuated in P2X4 KO mice. A P2 agonist ATPγS increased NLRP3 inflammasome signaling (NLRP3 and caspase 1 induction and IL-1β processing) in isolated renal proximal tubule cells from WT mice whereas these increases were absent in renal proximal tubules isolated from P2X4 KO mice. Moreover, 5-BDBD attenuated ATPγS induced NLRP3 inflammasome induction in renal proximal tubules from WT mice. Finally, P2X4 agonist ivermectin induced NLRP3 inflammasome and pro-inflammatory cytokines in cultured human proximal tubule cells. Taken together, our studies suggest that renal proximal tubular P2X4 activation exacerbates ischemic AKI and promotes NLRP3 inflammasome signaling.