Gentamicin is an effective aminoglycoside antibiotic drug but unfortunately up to 30% of gentamicin-treated patients may develop nephrotoxicity. Suggested mechanisms of gentamicin-induced nephrotoxicity included inflammation and oxidative stress injury. Evening primrose oil (EPO) has anti-inflammatory and antioxidant effects. The aim of current study was to assess the possible renoprotective effect of EPO/vitamin E combination treatment on gentamicin-induced nephrotoxicity in rats. Additionally, to address the responsible mechanism(s) of this effect. Eighteen adult male albino Sprague-Dawley rats were equally and randomly divided into three groups; normal control, gentamicin control and EPO/vitamin E treated groups. Gentamicin control group and EPO/vitamin E treated group received IP gentamicin injections for 5 days (100 mg/kg). EPO/vitamin E treated group received EPO/vitamin E (10 g and 200 IU /kg/day respectively orally). Renal function, oxidative stress and histopathological changes were assessed. Renal tissue expressions of tumor necrosis factor α (TNF α) and nuclear Factor κβ (NF- κβ) were assayed. Significant improvements of kidney function markers and tubular necrosis in EPO/vitamin E treated group were observed. EPO/vitamin E treatment significantly ameliorated the gentamicin-induced increase of renal lipid peroxidation and renal tissue expression of TNF α and NF-κβ. EPO/vitamin E treatment significantly ameliorated the gentamicin-induced decrease of renal glutathione and superoxide dismutase concentrations. Conclusion of current study is EPO/vitamin E combination treatment has renoprotective effect on gentamicin-induced nephrotoxicity by inhibiting renal TNF α /NF-κβ signaling pathway and the oxidative stress. Further researches for addressing the renoprotective effect of EPO treatment only and to determine other possible responsible mechanisms are needed.