Introduction: Iron and copper are absorbed in their reduced forms (ferrous and cuprous, respectively). Duodenal cytochrome b (Dcytb) functions as an intestinal ferric reductase; however, the nature of the putative intestinal cupric reductase remains unclear. We have shown previously that cells over-expressing Dcytb show not only enhanced ferric reductase activity but also display significant cupric reductase activity, suggesting that Dcytb may function as a dual metallo-reductase in intestinal enterocytes (Wyman et al FEBS Lett 2008, 582:1901-6). The aims of this study were to investigate whether intestinal Dcytb expression was regulated by copper, and whether Dcytb might play a role in cellular copper uptake. Methods and Materials: Human intestinal epithelial cells (Caco-2 and HuTu-80) were exposed to either the copper chelator TETA (0.5 mM) or copper (50 µM) for 24 h. Cells were harvested for RNA and expression of Dcytb and the copper transporter Ctr1 determined by qPCR. To determine whether Dcytb plays a role in copper uptake we employed a MDCK cell line which over-expresses Dcytb under the control of a tetracycline-repressible promoter (TET-Off MDCK, Clontech; Wyman et al 2008). Copper uptake was measured by ICP-OES. Results: In Caco-2 cells, Ctr1 expression was significantly increased by TETA treatment (p< 0.01) and significantly decreased by exposure to high copper medium (p<0.05). Basal Dcytb expression in Caco-2 cells was below the detection threshold in our studies so we used HuTu-80 cells as an alternative intestinal model. Dcytb expression was significantly increased in TETA-treated cells (p<0.01). Copper treatment did not significantly alter Dcytb mRNA compared with untreated controls. Copper uptake in Dcytb-overexpressing MDCK cells was significantly increased compared with untransfected control cells (+6.7-fold; p<0.01). Treatment with doxycycline (20ng/ml) significantly reduced copper uptake to levels seen in untransfected cells (p<0.01). Conclusion: These data demonstrate that Dcytb is a copper-regulated gene and that it plays a role in copper uptake. Taken together with our previous work we hypothesize that Dcytb functions as an intestinal cupric reductase providing Cu+ to Ctr1 for cellular uptake. This work forges a further link between mammalian iron and copper metabolism.