HFE2 (hemochromatosis type 2 gene) is highly expressed in skeletal muscle and liver hepatocytes. Its encoded protein, hemojuvelin (HJV), is a co-receptor for the bone morphogenetic proteins 2 and 4 (BMP2 and BMP4) and enhances the BMP-induced hepcidin expression. Hepcidin is a central iron regulatory hormone predominantly secreted from hepatocytes. HJV also binds neogenin, a membrane protein widely expressed in many tissues. Neogenin is required for the processing and release of HJV from cells. The role that neogenin plays in HJV trafficking was investigated, using HepG2 cells, a human hepatoma cell line. Knockdown of endogenous neogenin markedly suppresses HJV release but has no evident effect on HJV trafficking to the plasma membrane. The addition of a soluble neogenin ectodomain to cells markedly inhibits HJV release, indicating that the HJV shedding is not processed before trafficking to the cell surface. At the plasma membrane it undergoes endocytosis in a dynamin-independent but cholesterol-dependent manner. The additional findings that HJV release is coupled to lysosomal degradation of neogenin and that cholesterol depletion by filipin blocks both HJV endocytosis and HJV release suggest that neogenin-mediated HJV release occurs after the HJV-neogenin complex is internalized from the cell surface.