Gentamicin is an aminoglycoside antibiotic widely used for the treatment of life-threatening infections caused by Gram-negative bacteria. The use of gentamicin was limited due to its ototoxic and nephrotoxic adverse effects. The current study was designed to evaluate the protective effect of ethyl acetate fraction from Rotula aquatica (EFRA) against gentamicin induced nephrotoxicity. The antioxidant enzymes status, lipid peroxidation, nitrate and ROS level, serum markers like creatinine, Urea, BUN were estimated in the present study. The histopathological analysis of renal tissues was done by H&E and PAS staining. The mRNA level expression of KIM-1, NF-κB, TNF- α, and IL-6 were measured by semi-quantitative reverse transcription-polymerase chain reaction. The changes in antioxidant parameters were restored by the treatment of EFRA at different dose (50 mg/kg bwt, 100 mg/kg bwt). The serum parameters, ROS, MDA and nitrate level were decreased by administration of EFRA. The EFRA ameliorates histological changes associated with gentamicin induced nephrotoxicity. The mRNA level expression of KIM-1, NF-κB, TNF- α, and IL-6 were downregulated in EFRA treated groups. The results from present study reveals the role of EFRA as good anti-inflammatory and nephro protective drug.