In the present study, we investigated the protective effect of Withania somnifera, an indigenous medicinal herb used in ayurvedic traditional systems for more than 3000 years in India, on gentamicin (GEN)-induced nephrotoxicity. The root extract of three different doses of W. somnifera (viz., 250, 500, and 750 mg/kg) was administered orally to rats for 14 days before GEN treatment and thereafter concurrently with GEN (100 mg/kg) for 8 days. Nephrotoxicity was evident in GEN-treated rats by significant increase in kidney weight, urea, creatinine, urinary protein, and glucose, and significant reduction in body weights and potassium, which was histopathologically confirmed by tubular necrosis. In contrast W. somnifera (500 mg/kg) significantly reversed these changes as evidenced microscopically when compared to other two doses of W. somnifera (250 and 750 mg/kg), and there were no significant changes in the levels of sodium in the experimental animals compared to control. Thus, our results suggested the nephroprotective effect of Withania somnifera, which could be by enhancing antioxidant activity with natural antioxidants and scavenging the free radicals.