Diabetic nephropathy (DN) is the most common complication of type II diabetes. Rehmannia glutinosa (RG) is a traditional Chinese herb widely used in the treatment of diabetes and its complications; however, its pharmacological mechanism of action is incomplete. This study analysed the chemical composition of RG water extract (RGW) and RG ethanol extract (RGE) using UPLC-Q-TOF/MS and evaluated the in vitro activity. Moreover, a Sprague-Dawley rat model of DN was constructed using a high-fat diet and intraperitoneal injections of streptozotocin, followed by treatment of DN rats with RGW (250 and 500 mg/kg body weight) and observation of treatment effects. Water extraction of RG was efficient and had high total flavonoid and total polysaccharide contents. We identified 60 and 62 compounds from RGW and RGE, respectively, with RGW having the highest catalpol content. The combined antioxidant capacity of RGW was stronger than that of RGE, and the in vitro inhibitory activities of α-amylase and α-glucosidase were relatively high. RGW treatment resulted in reduced food intake, water intake, and blood glucose levels in DN rats. Furthermore, RGW improved blood lipid levels, improved kidney and hepatic function and antioxidant capacity, reduced inflammatory factor levels in DN rats, and improved pathological damage to the kidneys. The mRNA and protein expression levels of toll-like receptor 4, myeloid differentiation factor 88, and nuclear factor-κB p65 were all decreased. These results indicate that RG may be a potential therapeutic agent for improving DN, and its mechanism of action has been further clarified.