The aim of the study was to investigate the nephrotoxic effects of gentamicin in adult male sheep, and to identify the earliest signs of toxicity and the extent of clinical and biochemical changes. Twenty clinically healthy yearling male Iranian fattailed sheep were injected with gentamicin sulfate at a daily dose of 80 mg/kg for 9-10 d when nephrotoxicosis was induced. Blood samples were collected weekly before and after induction of nephrotoxicosis. Gentamicin-induced nephrotoxicity was characterised by increased creatinine and urea levels in serum, electrolyte imbalances, occurrence of albuminuria, and renal dysfunction. Significant elevation in respiratory and heart rates were observed one week after treatment (P < 0.05). There was a noticeable increase in water consumption, lethargy, and loss of appetite in treated sheep. There were significant correlations between serum creatinine and potassium (P = 0.004, r = 0.759), sodium (P = 0.017, r = 0.501), and urea (P = 0.021, r = 0.617) levels. Additionally, significant negative correlations between serum total protein and albumin and creatinine (P = 0.023, r = -0.484) and urea (P = 0.036, r = -0.381) were found. At necropsy, the kidneys were pale, swollen, and wet on the cut surface, especially perirenal tissues and ureters were oedematous. These findings confirmed the previous reports in other species.