Duodenal cytochrome B (Dcytb) is localized principally in the apical membrane of the enterocyte. It is thought to act as a ferric reductase that furnishes Fe(II), the specific and selective iron species transported by divalent metal transporter 1 (DMT1) in the duodenal enterocytes. Expression of both genes is strongly iron regulated and is thought to be required for transcellular iron trafficking in concert in response to physiological requirements. We tested this hypothesis by expressing Dcytb in Caco-2 cells, a human cell line model often used to mimic intestinal enterocytes. Iron uptake (59Fe) was significantly higher in Dcytb-transfected Caco-2 cells than in cells transfected with empty vector as a control. Fe(III) reductase activity of Dcytb was measured with ferrozine, a strong chelator of Fe(II) species. Cells expressing Dcytb exhibited enhanced ferric reductase activity as well as increased 59Fe uptake compared with cells transfected with empty vector as a control. Ferrozine blocked iron uptake and preincubation of cells with dehydroascorbate (to increase cellular ascorbate levels) stimulated iron uptake. Cotransfection of Dcytb and DMT1 resulted in an additive increase in iron uptake by the cells. The results confirm Dcytb can act as a ferric reductase that stimulates iron uptake in Caco-2 cells.