Diabetic nephropathy (DN) is one of the major causes of end-stage renal disease, and previously we demonstrated that NALP3 inflammasome was involved in the pathogenesis of DN. Here we investigated the mechanisms of NALP3 inflammasome activation in podocyte injury during DN. We found that, besides the activation of NALP3 inflammasome and upregulated thioredoxin-interacting protein (TXNIP), the glomerular expression of gp91 (phox) , a subunit of NADPH oxidase, was enhanced in DN mice simultaneously. Inhibiting NADPH oxidase abrogated NALP3 inflammasome activation, and IL-1β production and eventually protected podocytes from high glucose- (HG-) induced injury. TXNIP, an inhibitor of thioredoxin, acts as a suppressor for antioxidant defense system. Our observation indicated that in HG-exposed podocytes genetic deletion of TXNIP by shRNA reversed gp91 (phox) overexpression and alleviated the injury of podocyte. Collectively, our findings proposed that HG-induced NADPH oxidase activation was driven by TXNIP which subsequently triggered NALP3 inflammasome activation in podocytes and ultimately led to podocyte injury, and blocking TXNIP/NADPH oxidase signaling may be a promising treatment for DN.