Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an important coenzyme involved in cellular biosynthetic and redox metabolism. It has been recognized for its role in regulating neuroinflammation through coordinating redox reactions. Whether there are new actions other than redox regulation remain unclear. In this study we investigated a novel mechanism by which NADPH regulated microglia-mediated neuroinflammation. We showed that NADPH application significantly alleviated NLRP3 inflammasome activation in microglia and exerted neuroprotective effects both in vitro and in vivo neuroinflammation models. With P2X7R knockdown microglial cells and P2X7R mice, we demonstrated that P2X7R was a crucial mediator of the anti-inflammatory effects for the supplemented NADPH. We conducted whole-cell recording from murine microglial cell line BV2 cells, and found that application of ATP (1 mM) elicited an inward current, which was reduced by co-application of P2X7R antagonist A-438079 (20 μM) or NADPH (1 mM). By performing a drug affinity responsive targets stability (DARTS) assay, we revealed that NADPH (not NADP or NADH), like the P2X7R agonist ATP, bound to the extracellular domain of P2X7R, leading to the suppression of ATP-induced P2X7R activation. Our research provides the first evidence of NADPH as an endogenous inhibitor of P2X7R in modulation of microglia-mediated neuroinflammation. This study expands the biological functions of NADPH and offers a novel target for NADPH-based therapies in neuroimmune-related diseases.