Abstract: Gentamicin (GM) is a widely used antibacterial antibiotic however its optimal clinical benefit is limited by its serious nephrotoxicity and ototoxicity. The present study was aimed to investigate the influence of Thymoquinone (TQ), a compound isolated from Nigella sativa plant with predominant antioxidant property on a rat model of GM-induced nephrotoxicity. Four groups of adult male Wistar Albino rats (n = 10 per group) were treated for 10 consecutive days as follow: group 1; received normal saline and served as normal controls, group 2; received TQ (10 mg/kg/day; orally), group 3; received GM (80 mg/kg/day; intraperitoneal injection), and group 4; concurrently received GM + TQ. At day 11, the animals were sacrificed and the following parameters were evaluated: the levels of serum Creatinine (Cr) and Blood Urea Nitrogen (BUN) as biomarkers of renal function, the renal content of Thiobarbituric Acid Reactive Substances (TBARS) as an index of lipid perioxidation and oxidative stress total Glutathione (GSH) content and Glutathione S-Transferase (GST) activity in renal tissues as indices of antioxidant mechanisms and histopathological examination of kidney specimens. The biochemical results showed that GM administration induced nephrotoxicity associated with significant increases in serum levels of Cr, BUN and in renal content of TBARS with significant reductions in the renal GSH level and GST activity. The histopathological findings supported the presence of seriously injured kidney. However, concomitant administration of TQ efficiently reduced the development of GM nephrotoxicity and its associated biochemical and histopathological features. In conclusion, these data prove that TQ mediates; via, at least in part, its antioxidant property, a marked renoprotective effect against GM-induced nephrotoxicity in rats.