Juvenile hemochromatosis is an iron overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP) 1 and hemojuvelin (HFE2)2. We have previously shown that hemojuvelin is a bone morphogenetic protein (BMP) co-receptor and that BMP signals regulate hepcidin expression and iron metabolism3 , 4. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here, we show that in vitro, compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc more potently inhibits hepcidin induction by BMP-2 or BMP-4, but less potently inhibits BMP-6. In vivo, HJV.Fc or a neutralizing BMP-6 antibody inhibits hepcidin expression and increases serum iron, while DRAGON.Fc has no effect. Notably, Bmp6 null mice have a phenotype resembling hereditary hemochromatosis with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP-6, and we show that BMP-6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP-6 as a ligand for HJV and an endogenous regulator of hepcidin expression and iron metabolism in vivo.Secreted by the liver 5 , hepcidin inhibits intestinal iron absorption and macrophage iron release by decreasing cell surface expression of the iron exporter ferroportin 6 . Hepcidin is upregulated by iron administration 5,[7][8] and inhibited by anemia 7 . Hepcidin deficiency and unchecked ferroportin activity are the common pathogenic mechanisms underlying the genetic iron Figure 1a, DRAGON.Fc significantly inhibited hepcidin promoter induction by BMP-2 or BMP-4, but was less effective in inhibiting BMP-5, BMP-6, or BMP-7, and did not inhibit BMP-9. In comparison with HJV.Fc, DRAGON.Fc was significantly more potent against BMP-2 (Fig. 1b) and BMP-4 (Fig. 1c), but was less potent against BMP-6 (Fig. 1d). DRAGON.Fc also inhibited endogenous hepcidin mRNA expression in hepatoma-derived HepG2 cells, where basal hepcidin expression is dependent in part on endogenous BMP-2, BMP-4, and BMP-6 ligands 4 ( Supplementary Fig. 1).We then tested whether DRAGON.Fc administration affected hepcidin expression and iron metabolism in vivo. HJV.Fc at a similar dose was used as a positive control. In contrast to HJV.Fc, DRAGON.Fc had no effect on hepatic hepcidin mRNA (Fig. 1e), splenic ferroportin ( Supplementary Fig. 2a-b.), serum iron ( Fig. 1f), serum transferrin saturation, liver iron content, or spleen iron content (Supplementary Fig. 2c-e) compared with mock treated control mice. Anti-BMP-2 activity in the serum of DRAGON.Fc treated mice was confirmed by the ability of this serum to inhibit BMP-2 induction of hepcidin promoter activity in vitro compared with serum from mock treated mice ( Supplementary Fig. 2f).Since DRAGON.Fc had no effect in vivo despite its higher potency in vitro as an inhibitor of BMP-2 and BMP-4 compared with HJV.Fc, and since DRAGON.Fc was less potent at inhibiting BMP-6 compared with HJV.Fc, we hypothesized that the BMP-6 inhibiting p...