Background: Gentamicin (GNT) is a highly effective aminoglycoside antibiotic that is commonly used to treat life-threatening bacterial infections. Aim: The aim of the current study is to determine whether tadalafil can protect against GNT-induced nephrotoxicity. Methods: Twenty-four male Albino rats were used in the study, which were randomly divided into four groups, six animals each. Control untreated group received distilled water (5ml/kg, P.O) for 12 days and on days 6-12, they received (5ml/kg,i.p) normal saline daily, one hour after oral administration of distilled water. Tadalafil group received tadalafil (5mg/kg, P.O) for 12 days and on days 6-12, they received (5ml/kg,i.p) of normal saline daily, one hour after oral administration of tadalafil. Gentamicin group received distilled water (5ml/kg, P.O) and on days 6-12, they received gentamicin (100mg/kg,i.p) one hour after oral administration of distilled water. Gentamicin+Tadalafil group received tadalafil (5mg/kg, P.O) for 12 days and on days 6-12, they received gentamicin (100mg/kg,i.p) one hour after oral administration of tadalafil. Body weight and kidney weight were investigated. Urine volume as well as urinary albumin, creatinine, creatinine clearance and albumin /creatinine ratio (ACR) were evaluated. Besides, serum levels of urea and creatinine were measured as kidney function parameters. Renal tissues oxidative stress markers as malondialdehyde (MDA) and the antioxidant enzymes as superoxide dismutase (SOD) and catalase (CAT) activities. Renal cortex nitric oxide (NO) and kidney injury molecule-1 (KIM-1) were evaluated. Histological analysis and immunohistochemical expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) were performed. The results of the present study showed that GNT decreased creatinine clearance and increased the serum levels of renal function parameters. GNT caused a significant increase in renal cortex MDA, NO and urine levels of KIM-1, while it decreased CAT and SOD activities.  Gentamicin   administration resulted in increased immunohistochemical expression of iNOS enzyme while decreasing eNOS expression. Renal corpuscles and tubules also showed histological and ultrastructural changes. Pretreatment with tadalafil, on the other hand, reversed the effects of GNT administration.  In conclusion, findings of the present study indicate that tadalafil reduces GNT-induced kidney damage by inhibition of inflammation, oxidative stress, and apoptosis.