We investigated the protective effect of Withania somnifera root powder (used in ayurvedic medicine in India) on gentamicin (GEN) induced nephrotoxicity in male Wistar rats. The root powder was administered orally to rats for 14 days before GEN treatment and thereafter with GEN for 8 days. Nephrotoxicity was manifested in GEN-treated rats as significant increases in urea, creatinine, uric acid, non protein nitrogen, urinary protein, N-acetyl-beta-D-glucosaminidase, thiobarbituric acid reactive substances, hydroperoxides, followed by a significant reduction in glutathione peroxidase, superoxide dismutase, catalase, and reduced glutathione in liver and kidney tissues, histopathologically confirmed by tubular necrosis. W. somnifera treatment altered the antioxidant status and significantly reversed the levels as seen microscopically. The results show that the root powder of W. somnifera with the presence of natural antioxidants, bioflavanoids, and other bioactive compounds scavenged the free radicals generated by GEN and ameliorated the severity of GEN-induced nephrotoxicity by enhancing the antioxidant system and protecting the cellular integrity of kidney and liver tissues.