Gentamicin is commonly used against gram-negative microorganisms. Its therapeutic use is mainly limited by nephrotoxicity. This study was aimed at evaluating the effect of rutin on oxidative stress, inflammation, apoptosis, and autophagy in gentamicin-induced nephrotoxicity in rats. The rats were treated with saline intraperitoneally (group I), 150 mg/kg of rutin orally (group II), 80 mg/kg of gentamicin intraperitoneally for 8 d (group III), or 150 mg/kg of rutin plus 80 mg/kg of gentamicin (group IV). The serum urea, creatinine, kidney malondialdehyde (MDA), and reduced glutathione (GSH) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity and protein concentration were measured, and renal histopathology analysis and immunohistochemical staining were performed. Rutin pretreatment attenuated nephrotoxicity induced by gentamicin by reducing the urea, creatinine, and MDA levels and increasing the SOD, CAT, and GPx activity, and the GSH levels. The rutin also inhibited inducible nitric oxide synthase (iNOS), cleaved caspase-3 and light chain 3B (LC3B), as evidenced by immunohistochemical staining. The present study demonstrates that rutin exhibits antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic effects and that it attenuates gentamicin-induced nephrotoxicity in rats.