This study was aimed to evaluate the protective effect of extra virgin olive oil (EVOO) and its fractions (hydrophilic (HF) and lipophilic (LF)) treatment on 2,4-dichlorophenoxiacetic acid (2,4-D)-induced nephrotoxicity in rats. The Wistar rats (n ¼ 80, 210-230 g) were divided into eight groups having ten animals, i.e., control group (C), (2,4-D) group that received 2,4-D (5 mg/kg b.w.), (2,4-D/EVOO) group was treated with 2,4-D plus EVOO, (2,4-D/olive oil hydrophilic fraction, OOHF) group that received 2,4-D plus HF, (2,4-D/olive oil lipophilic fraction, OOLF) group was treated with 2,4-D plus LF, (EVOO) group that received only EVOO, (OOHF) group was given HF and (OOLF) group was treated with the LF. These components were administered daily by gavage for 4 wk. 2,4-D administration affected plasma urea and creatinine levels, which increased while uric acid significantly decreased. A marked increase of malondialdehyde level and with a significant decrease in enzymatic antioxidant's activities, were also observed in 2,4-D treated rats. The co-administration of EVOO and its fractions along with 2,4-D resulted in a reversal of 2,4-D induced biochemical changes in kidney accompanied by a significant decrease in lipid peroxidation and an increase in the level of the antioxidant defence system. Light microscopy investigations revealed that 2,4-D exposure induced numerous histopathological alterations in the kidney. EVOO administration with 2,4-D severely reduced the toxicity of 2,4-D and preserved the normal histological architecture of the renal tissue. However, a milder histopathological improvement was observed in animals co-treated with HF or LF plus 2,4-D. In conclusion, the present study suggest that the nephroprotective potential of EVOO against 2,4-D toxicity might be due to the synergic effect of the two fractions, which could be useful for achieving optimum effects in 2,4-D-induced renal damage.