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The kidney is a typical organ undergoing age and injury. Hyperoside is reported to be useful for preventing aging induced by D-galactose (D-gal). However, therapeutic mechanisms remain unclear. We thereby aimed to verify whether hyperoside, compared to vitamin E (VE), could alleviate renal aging and injury by regulating autophagic activity and its related signaling pathways. In vivo, rats were administered with either hyperoside or VE after renal aging modeling induced by D-gal. Changes in renal aging and injury markers, autophagic activity and AMPK-ULK1 signaling pathway in the kidneys were analysed. In vitro, the NRK-52E cells exposed to D-gal were used to investigate regulative actions of hyperoside and VE on cell viability, renal tubular cellular aging markers, autophagic activity and its related signaling pathways by histomorphometry, immunohistochemistry, immunofluorescence, lentiviral transfection and Western blot. Aging and injury in the kidneys and renal tubular cells induced by D-gal were ameliorated by hyperoside and VE. Hyperoside and VE inhibited autophagic activity through mTOR-independent and AMPK-ULK1 signaling pathways. Hyperoside, as a component of phytomedicine similar to VE, attenuated renal aging and injury induced by D-gal via inhibiting AMPK-ULK1-mediated autophagy. This study provides the first evidence that hyperoside contributes to the prevention of age-associated renal injury.