This study was designed to evaluate the protective effects of selenium (Se) supplementation in minimizing 5-fluorouracil (5-FU) induced lipid peroxidative damage of the small intestine in rats. Rats were fed a nonpurified diet, with (Se group) or without (N group) supplementation of 2.0 ppm sodium selenite in drinking water for 5 weeks. 5-FU (150 mg/kg) was administrated intraperitoneally. Malondialdehyde (MDA) level, glutathione peroxidase (GSH-Px) activity, myeloperoxidase (MPO) activity, disaccharidase activity, Se content, and intestinal permeability were determined before and on days 1, 3, and 5 after the injection of 5-FU. Se content in liver and jejunal mucosa was significantly higher in the Se group than in the N group at different times. MDA concentration in blood, jejunal mucosa, liver, kidney, and heart rate was significantly higher in the N group than in the Se group on day 3, whereas it also was significantly higher in blood and jejunal mucosa in the N group than in the Se group on day 1. Erythrocyte GSH-Px activity was significantly higher in the Se group than in the N group at different times. GSH-Px activity in jejunal mucosa on day 1 and in heart, liver, and kidney on day 3 was significantly higher in the Se group than in the N group. Activities of jejunal sucrase and maltase were significantly higher in the Se group than in the N group on day 3. Intestinal MPO activity was significantly higher in the N group than in the Se group on day 1. There was no significant difference in intestinal permeability and water content between the two groups. The results indicate that supplementation of Se may protect against lipid peroxidative damage of the small intestine induced by 5-FU.