Hepcidin (Hamp1) is a liver-expressed iron-regulatory hormone that controls systemic iron homeostasis. Hepcidin is in turn regulated by the Bone Morphogenetic Protein (BMP)/SMAD family member (Smad) signaling pathway. Bmp6 expression in hepatocytes is up-regulated by iron and signals through phosphorylation of Smad1/5/8 to induce hepcidin. The hemochromatosis-associated proteins Hfe and Transferrin Receptor 2 (Tfr2) are known to be upstream regulators of hepcidin, although their precise roles are still unclear. To investigate this, we assessed regulatory responses to iron loading via dietary or parenteral routes in wild type and Hfe-/-/Tfr2-/- mice. Dietary iron accumulated exclusively in hepatocytes, while parenteral iron accumulated predominantly in non-parenchymal cells. The hepcidin regulatory response to both challenges was significantly diminished in the Hfe-/-/Tfr2-/- mice, however, regulation of Hamp1 and Bmp6 differed between the two. In Hfe-/-/Tfr2-/- mice, Bmp6 up-regulation was dampened in response to iron feeding, but not to injected Fe-dextran. Phosphorylation of Smad1/5/8 (pSmad1/5/8) was unaffected relative to Bmp6 in all animals. Hfe-/-/Tfr2-/- mice fed iron showed a 59% reduction in Hamp1 relative to pSmad1/5/8, while Fe-dextran injected Hfe-/-/Tfr2-/- mice showed a 96% reduction in Hamp1 relative to pSmad1/5/8 levels. Finally, regulation of Bmp6 in Hfe-/- and Tfr2-/- single null mice was compared. Tfr2-/- mice had a blunted Bmp6 response equal to that of Hfe-/-/Tfr2-/-, while Hfe-/- mice showed no loss of Bmp6 regulation. In conclusion, this study highlights differential roles of Hfe and Tfr2 in the regulatory responses to iron administered by different routes and distributed in different cell types. Significantly, it shows that Tfr2, but not Hfe, is required for hepatocyte iron-induced up-regulation of Bmp6, and suggests that Hfe asserts its regulatory influence on Hamp1 expression downstream of Smad1/5/8.