Pyruvate is an endogenous antioxidant and anti-inflammatory substance. The present study was implemented to investigate the protective effect of ethyl pyruvate (EP) against the development and progression of diabetic nephropathy in an in vivo and in vitro model. Diabetic rats were prepared by injecting streptozotocin (65 mg/kg). Those that developed diabetes after 72 h were treated with EP (40 mg/kg) intraperitoneally. Diabetic rats without pyruvate treatment and nondiabetic rats were used for control. As an in vitro experiment, rat mesangial cells cultured primarily from Sprague-Dawley rats were treated in high-glucose (HG; 50 mM) or normal-glucose (NG; 5 mM) conditions and with or without pyruvate. Pyruvate-treated diabetic rats exhibited decreased albuminuria and attenuated NADPH-dependent reactive oxygen species generation. Immunohistochemistry showed reduced laminin, type IV collagen, and fibronectin deposition in the glomeruli compared with nontreated diabetic rats. Parallel changes were shown in tissue mRNA and protein expression levels of monocyte chemoattractant protein-1, transforming growth factor-β1, laminin, fibronectin, and type IV collagen in the kidney. Concordantly, protective effects were also exhibited in the mesangial cell culture system. These findings suggest that pyruvate protects against kidney injury via NADPH oxidase inhibition. The present study established that activation of NADPH oxidase plays a crucial role in diabetes-induced oxidative stress, glomerular hypertrophy, and ECM molecule expression. Pyruvate exhibited a renoprotective effect in the progression of experimental diabetic nephropathy. Future research is warranted to investigate the protective mechanism of pyruvate more specifically in relation to NADPH oxidase in diabetic nephropathy.