The protective role of diallyl sulfide (DAS) in attenuating gentamicin-induced nephrotoxicity has been reported earlier. However, the mechanism of induction of antioxidants by DAS in nephrotoxicity remains elusive. This study is aimed to elucidate the role of a transcription factor, Nuclear factor E2-related factor 2 (Nrf2) in inducing antioxidants and phase II enzymes during gentamicin toxicity in Wistar rats. DAS was administered intraperitoneally at a dosage of 150 mg/kg body weight once daily for 6 days. Gentamicin was administered intraperitoneally at a dosage of 100 mg/kg body weight, once daily for 6 days. Gentamicin-induced rats showed a significant increase in the levels of kidney markers and the activities of urinary marker enzymes, which was reversed upon treatment with DAS. A significant increase in kidney myeloperoxidase (MPO) and lipid peroxidation (LPO) levels was observed in gentamicin-induced rats, which was reduced upon treatment with DAS. Gentamicin-induced rats also showed a significant decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and quinone reductase (QR) in rat kidney, which was increased upon treatment with DAS. Immunohistochemical studies in gentamicin-induced rats demonstrated a marked increase in the immunoreactivity of inducible nitric oxide synthase (iNOS), nuclear transcription factor (NF-kappaB) and tumor necrosis factor alpha (TNF-alpha) that were reduced after treatment with DAS. Further, the involvement of Nrf2 in antioxidant induction was analyzed by Western blot and immunofluorescence. To conclude, DAS enhances antioxidants and suppresses inflammatory cytokines through the activation of Nrf2, thereby protecting the cell against oxidative stress induced by gentamicin.