Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. Natural flavonoids, as rutin, are well-known antioxidants, and could be efficient protective agents. Therefore, the present study was undertaken to evaluate the protective influence of rutin supplementation to improve rat antioxidant systems against IOL-induced hepatic oxidative stress. Sixty male albino rats were randomly divided to three equal groups. The first group, the control, the second group, iron overload group, the third group was used as iron overload+rutin group. Rats received six doses of ferric hydroxide polymaltose (100 mg/kg b.w.) as one dose every two days, by intraperitoneal injections (IP) and administerated rutin (50 mg/kg b.w.) as one daily oral dose until the sacrificed day. Blood samples for serum separation and liver tissue specimens were collected three times, after three, four and five weeks from the onset of the experiment. Serum iron profiles {total iron, total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), transferrin (Tf) and transferrin saturation % (TS%)}, ferritin, albumin, total Protein, total cholesterol, triacylglycerols levels, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined. Moreover, total iron in the liver, L-malondialdehyde (L-MDA), glutathione (GSH), nitric oxide (NO) and total nucleic acid (TNA) levels and glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) activities were also determined. The obtained results revealed that, iron overload (IOL) resulted in significant increase in serum iron, TIBC, Tf, TS% and ferritin levels and AST and ALT activities, and also increased liver iron, L-MDA, and NO levels. Meanwhile, it decreased serum UIBC, total cholesterol, triacylglycerols, albumin, total protein and liver GSH, TNA levels, and Gpx, CAT and SOD activities when compared with the control group. Rutin administration to iron-overloaded rats resulted in significant decrease in serum total iron, TIBC, Tf, TS%, ferritin levels, and AST and ALT activities and liver total iron, L-MDA, and NO levels with significant increases in serum UIBC, albumin, total protein and total cholesterol levels, and in liver GSH, CAT and SOD activities compared with the IOL group. This study provides in vivo evidence that rutin administration can improve the antioxidant defence systems against IOL-induced hepatic oxidative stress in rats. This protective effect in liver of iron-loaded rats may be due to both antioxidant and metal chelation activities.