Ethnopharmacological relevance: Ginkgo biloba extract (GbE) is derived from a medicinal plant and suggested as a treatment for diabetic nephropathy (DN), but the mechanism was not clarified. Aim of study: The present study investigated whether GbE prevented DN via activation of heme oxygenase (HO)-1. Materials and methods: Streptozotocin-induced diabetic mice were fed a high-fat diet to generate DN. Human and murine podocytes were used for the in vitro study. Results: GbE improved renal function via decreasing glomerular hypertrophy, the kidney/body weight ratio, and albuminuria in DN mice. GbE reversed the reduction of synaptopodin and nephrin and enhanced HO-1 expression in the kidneys of DN mice. GbE decreased the enhancement of TNF-α, IL-6, fibronectin, and lipid accumulation in the glomeruli of DN mice. GbE attenuated the uptake of oxidized low-density lipoprotein and reduced the production of ROS in high glucose-stimulated podocytes, and HO-1 inhibitor treatment abrogated the protective effects of GbE. Nuclear factor erythroid 2-related factor 2 (Nrf-2) siRNA significantly abolished the beneficial effects of GbE via decreased HO-1 expression and enhanced TNF-α and IL-6 levels. Conclusions: GbE protected podocytes against hyperglycemia and prevented the development of DN via Nrf-2/HO-1 activation. Our findings provide further mechanistic insight into the potential use of GbE in clinical DN.