The effect of the putative iron regulatory peptide hepcidin on iron absorption was investigated in mice. Hepcidin peptide was synthesized and injected into mice for up to 3 days, and in vivo iron absorption was measured with tied-off segments of duodenum. Liver hepcidin expression was measured by reverse transcriptase-polymerase chain reaction. Hepcidin significantly reduced mucosal iron uptake and transfer to the carcass at doses of at least 10 microg/mouse per day, the reduction in transfer to the carcass being proportional to the reduction in iron uptake. Synthetic hepcidin injections down-regulated endogenous liver hepcidin expression excluding the possibility that synthetic hepcidin was functioning by a secondary induction of endogenous hepcidin. The effect of hepcidin was significant at least 24 hours after injection of hepcidin. Liver iron stores and hemoglobin levels were unaffected by hepcidin injection. Similar effects of hepcidin on iron absorption were seen in iron-deficient and Hfe knockout mice. Hepcidin inhibited the uptake step of duodenal iron absorption but did not affect the proportion of iron transferred to the circulation. The effect was independent of iron status of mice and did not require Hfe gene product. The data support a key role for hepcidin in the regulation of intestinal iron uptake.