Background: The present study was conducted to elucidate the protective role of Withania somnifera against bromobenzene induced nephrotoxicity and mitochondrial dysfunction in rats. Methods: In this study, Wistar albino rats of either sex were divided into six groups consisting of six animals each. The first one was control, those in group II received bromobenzene (10mmol/kg, intragastric intubation) once, but group III and IV animals received W. somnifera (250 and 500mg/kg, orally), respectively for 8 days followed by bromobenzene once on the 8th day and silymarin (100mg/kg, orally) was given for 8 days to group V animals and then bromobenzene on the last day. Group VI animals received only W. somnifera (500mg/kg) for 8 days. Results: The levels of renal lipid peroxidation, lysosomal enzymes and glycoprotein were increased significantly (p<0.05) in the bromobenzene alone treated rats and antioxidant status and mitochondrial enzymes were found to be decreased, when compared to the control group. The levels of kidney functional markers (urea, uric acid and creatinine) were also found to be abnormal in serum of bromobenzene alone treated rats. On the other hand, administration of W. somnifera (250 and 500mg/kg) along with bromobenzene offered a significant dose-dependent protection to the biochemical alterations as observed in the bromobenzene alone treated rats, which was also evidenced by histopathology. Conclusion: Thus, the oral administration of W. somnifera significantly protected against the bromobenzene induced nephrotoxicity and renal mitochondrial dysfunction in rats. © 2014 Informa Healthcare USA, Inc. All rights reserved: reproduction in whole or part not permitted.