Diabetic nephropathy (DN) is one of the most severe microvascular complications of diabetes and has become the leading cause of end-stage renal disease formation. The pathogenesis of diabetic nephropathy is very complex and is still not fully understood. Fisetin is a flavonoid polyphenolic compound that is widely found in different fruits, vegetables, and medicinal plants. Many studies have indicated that it has a variety of pharmacological activities. In this study, we investigated the mechanism of action of fisetin in the protection of DN-induced podocyte injury both in vivo and in vitro. Results showed that fisetin could reduce high glucose (HG)-induced podocyte injury and streptozotocin (STZ)-induced diabetic nephropathy in mice. According to the histopathological staining results, fisetin ameliorated DN-induced glomerular injury in a dose-dependent manner. Western blot and immunofluorescence results showed that fisetin effectively promoted the expression of podocyte functional integrity marker proteins and inhibited the expression of podocyte injury marker proteins. In addition, according to the Western blot and RT-qPCR results, fisetin activates the nuclear translocation of Nrf2 to exert antioxidative stress ability and affects the expression of downstream antioxidant enzymes HO-1, GPX4, and other ferroptosis-related markers, thereby protecting against HG-induced podocyte ferroptosis and oxidative stress injury in DN mice. In summary, this study demonstrated that fisetin could enhance the antioxidative stress capacity of DN mice by promoting the activation of the Nrf2/HO-1/GPX4 signaling pathway in renal tissues, and attenuated HG-induced podocytes injury and STZ-induced DN in mice.