Abstract: Background Hederagenin is a naturally occurring pentacyclic triterpenoid found in several medicinal plants traditionally used for treating renal and metabolic disorders. Its ability to mitigate renal senescence in diabetic kidney disease (DKD) and the associated epigenetic mechanisms have not yet been fully elucidated. Methods db/db mice were used in vivo to model DKD-associated renal senescence, while palmitic acid-treated human renal proximal tubular epithelial cells (HK-2 cells) were used in vitro as a senescence model. Renal injury, senescence, DNA damage, and Klotho expression were evaluated using histological, biochemical, and molecular analyses. GEO datasets derived from human DKD samples and corresponding controls were analyzed to assess Klotho expression. Integrated target-binding and methylation analyses were performed to examine the interaction between hederagenin and DNA methyltransferase 1 (DNMT1), as well as the effects of hederagenin on Klotho promoter methylation and DNMT1 occupancy. Results Hederagenin reduced renal senescence, fibrosis, and DNA damage in vivo and in vitro, while restoring Klotho expression, which was decreased in patient datasets and experimental models. Mechanistically, hederagenin directly bound DNMT1, diminished DNMT1 recruitment to the Klotho promoter, and attenuated aberrant promoter hypermethylation, thereby reactivating Klotho. Conclusion Hederagenin mitigates renal senescence in DKD through a DNMT1–Klotho DNA methylation axis, supporting its ethnopharmacological potential for DKD management.