Background Iron-induced oxidative stress in the small intestine may alter gene expression in the intestinal mucosa. The present study aimed to determine which genes are mediated by an iron-induced oxidative challenge in the human small intestine. Methods Eight healthy volunteers (22+/-2 y) were tested on two separate occasions, in a randomized cross-over design. After duodenal tissue sampling by gastroduodenoscopy, a perfusion catheter was inserted orogastrically to perfuse a 40-cm segment of the proximal small intestine with saline and, subsequently, with either 80 or 400 mg iron as ferrous gluconate. After the intestinal perfusion, a second duodenal tissue sample was obtained. Results Thiobarbituric acid reactive substances (TBARS), an indicator of lipid peroxidation, in intestinal fluid samples increased significantly and dose-dependently at 30 min after the start of perfusion with 80 or 400 mg iron, respectively (P<0.001). During the perfusion with 400 mg iron, the increase in TBARS was accompanied by a significant, momentary rise in trolox equivalent antioxidant capacity (TEAC), an indicator of total antioxidant capacity (P<0.05). The expression of eighty-nine gene reporters was significantly altered by both iron interventions. Functional mapping showed that both iron dosages mediated six distinct processes. Three of those processes involved G protein receptor coupled pathways. The other processes were associated with cell cycle, complement activation and calcium channels. Conclusion Iron administration in the small intestine induced, dose-dependent, lipid peroxidation and a momentary antioxidant response in the lumen, mediated the expression of at least 89 individual gene reporters, and affected at least six biological processes.