The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation, and is downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2^MASK), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2^MASK shows no cleavage activity and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2^MASK. The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.