Oxidative stress results from the imbalance between reactive oxygen species (ROS) production and antioxidant defence and is primarily involved in aging. The current study investigated the antioxidant activity of rutin in aging in rats induced by D-galactose (D-gal) for 42 days. Rutin was orally used at doses of 50 and 100 mg kg-1 daily. Results showed that D-gal induced oxidative alterations in the brain and liver recognized via upregulation of aging and oxidative markers. In contrast, rutin ameliorated the oxidative stress induced by D-gal by enhancing antioxidant markers such as superoxide dismutase-1, glutathione peroxidase-1, and glutathione S-transferase-α. Also, rutin significantly decreased the accumulation of β-galactosidase and reduced the expression of p53, p21, Bcl-2-associated X protein (Bax), caspase-3 (CASP3), and mammalian target of rapamycin (mTOR) in brain and hepatic tissues. Rutin potentially attenuated these aging-related oxidative alterations in a dose-dependent manner. Moreover, rutin markedly reduced the increased immunohistochemical expression of β-galactosidase, 8-hydroxy-2'-deoxyguanosine, calcium-binding adapter molecule 1, glial fibrillary acidic protein, Bax, and interleukin-6 and significantly increased Bcl2, synaptophysin, and Ki67. Furthermore, a molecular docking study revealed that rutin exhibited high affinity to rat and human caspases, PI3K/AKT/mTOR, and the IL-6 receptor. Finally, we can conclude that rutin supplementation can be a promising natural protective compound that could delay aging and maintain health.