An increasing number of investigations have revealed that podocytes play a crucial role in the development and progression of diabetic nephropathy (DN). Quercetin (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one) is the most common flavonol in the diet and is one of the most prominent dietary antioxidants, which might have a protective effect on DN. The present study was designed to investigate the protective effect of quercetin on podocyte impairment in a rat model of DN, as well as underlying molecular mechanisms. All diabetic rats were induced by a single intraperitoneal injection of streptozotocin, and quercetin was administered daily at a dose of 50 mg kg⁻¹ or 100 mg kg⁻¹ for 12 weeks. In the present study, quercetin markedly decreased blood glucose levels, kidney-to-body weight ratio, albuminuria, creatinine clearance rate, blood urea nitrogen, and triglycerides and significantly attenuated oxidative stress. Moreover, quercetin was observed to inhibit podocyte effacement and decrease the thickness of glomerular basement membranes. Mechanistically, quercetin significantly increased the expression of podocyte-specific markers nephrin and podocin and decreased expression of the podocyte injury marker desmin in DN rats. Quercetin also inhibited activation of the TGF-β1/Smad signaling pathway in DN rats by decreasing expression of TGF-β1, p-Smad2, and p-Smad3, and increasing Smad7 expression. These findings suggest that quercetin administration ameliorated podocyte injury in DN rats, possibly by inhibiting oxidative stress and the TGF-β1/Smad signaling pathway. Thus, quercetin may be manipulated to act as a potential drug for prevention of early diabetic nephropathy.