This work aims to evaluate the renoprotective effect of luteolin on expression of Nrf2 and miR320 in ischemia-reperfusion (I/R) injury in rats. Thirty rats were randomly divided into five groups; control, Luteolin (50 mg/kg), ischemia-reperfusion (I/R), DMSO (0/1%) + I/R and Luteolin+I/R, (n = 6 each). Administration of luteolin and DMSO was carried out by gavage for 3 days before renal I/R. Then, the rats were subjected to bilateral renal ischemia for 45 min and followed by reperfusion for 2 h. All rats were killed and the renal function, histological changes, oxidative stress degree, in all of groups were evaluated. In addition, the effects of luteolin on renal expression of Nrf2 and miR320 were examined by immunohistochemistry and real time- PCR. Luteolin significantly improved the creatinine (Cr) and blood urea nitrogen (BUN) levels in Luteolin + I/R group compared to I/R group (p < 0.001 and p < 0.001 respectively). Reduction of enzymatic activity of superoxide dismutase, glutathione peroxidase and catalase in I/R and DMSO + I/R groups, was significantly improved by Luteolin (p < 0.05) in Luteolin + I/R group. Pre-treatment with luteolin also resulted in significant reduction in tissue MDA level (p < 0.001), Nrf2 (p < 0.001) and miR320 expression (P < 0.05) that were increased by renal I/R. Also, the rats pretreated with luteolin had nearly normal structure of the kidney. These results indicate that luteolin protects the kidney against I/R injury via reducing oxidative stress, increasing antioxidant enzymes and reducing expression of Nrf2 and miR320.