Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77-82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis.