Hephaestin is a multicopper ferroxidase expressed mainly in the mammalian small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this study, the human hephaestin ectodomain was expressed in baby hamster kidney cells and purified to electrophoretic homogeneity. Ion exchange chromatography of purified recombinant human hephaestin (rhHp) resulted in the isolation of hephaestin fractions with distinct catalytic and spectroscopic properties. The fraction of rhHp with the highest enzymatic activity also showed an enhanced molar absorptivity at 600 nm, characteristic of type 1 copper sites. Kinetic analysis revealed that rhHp possesses both high-affinity and low-affinity binding sites for ferrous iron. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into rhHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type rhHp and mutants demonstrated the important roles of hephaestin residues E960 and H965 in the observed ferroxidase activity.