Gentamicin (GIN) is a widely used antibiotic, but nephrotoxicity restricts its application. GIN affects mainly renal tubules due to presence of megalin/cubulin complex, which is responsible for its uptake in proximal tubular epithelial cells. In this study, we investigated whether administration of GIN dose (100 & 120 mg/kg) once/day or fractionation of the dose over 4 hours could produce more nephrotoxic effect in rats. Kidney function was evaluated through assessment of serum creatinine (Cr), blood urea nitrogen (BUN), and glomerular filtration rate (GFR). Furthermore, these biochemical estimations were supported by the histopathological study. Administration of GIN 100 & 120 mg/kg once/day produced non-significant changes in kidney function biomarkers, in addition to preserved histological structure of glomeruli. However, fractionation of GIN dose 120 mg/kg over 4 hours/day produced significant deterioration of kidney function, as well as histopathological alterations. Fractionated dosing of GIN every day is more nephrotoxic than administration of the dose once/day. That could be attributed to saturation of megalin/cubulin complex and thus impaired endocytosis of GIN in proximal tubular epithelial cells when administered once daily. Thus, we suggest that administration of GIN in fractionated doses is more suitable for induction of nephrotoxicity in rats.