Hereditary hemochromatosis (HHC) is one of the most frequent genetic disorders in humans. In healthy individuals, absorption of iron in the intestine is tightly regulated by cells with the highest iron demand, in particular erythroid precursors. Cloning of intestinal iron transporter proteins provided new insight into mechanisms and regulation of intestinal iron absorption. The aim of this study was to assess whether, in humans, the two transporters are regulated in an iron-dependent manner and whether this regulation is disturbed in HHC. Using quantitative PCR, we measured mRNA expression of divalent cation transporter 1 (DCT1), iron-regulated gene 1 (IREG1), and hephaestin in duodenal biopsy samples of individuals with normal iron levels, iron-deficiency anemia, or iron overload. In controls, we found inverse relationships between the DCT1 splice form containing an iron-responsive element (IRE) and blood hemoglobin, serum transferrin saturation, or ferritin. Subjects with iron-deficiency anemia showed a significant increase in expression of the spliced form, DCT1(IRE) mRNA. Similarly, in subjects homozygous for the C282Y HFE mutation, DCT1(IRE) expression levels remained high despite high serum iron saturation. Furthermore, a significantly increased IREG1 expression was observed. Hephaestin did not exhibit a similar iron-dependent regulation. Our data show that expression levels of human DCT1 mRNA, and to a lesser extent IREG1 mRNA, are regulated in an iron-dependent manner, whereas mRNA of hephaestin is not affected. The lack of appropriate downregulation of apical and basolateral iron transporters in duodenum likely leads to excessive iron absorption in persons with HHC.