AbstractThe hepcidin‐ferroportin axis underlies the pathophysiology of many iron‐associated disorders and is a key target for the development of therapeutics for treating iron‐associated disorders. The aims of this study were to investigate the dynamics of hepcidin‐mediated ferroportin internalization and the consequences of a novel disease‐causing mutation on ferroportin function. Specific reagents for ferroportin are limited; we developed and characterized antibodies against the largest extracellular loop of ferroportin and developed a novel cell‐based assay for studying hepcidin‐ferroportin function. We show that hepcidin‐mediated ferroportin internalization is a rapid process and could be induced using low concentrations of hepcidin. Targeted next‐generation sequencing utilizing an iron metabolism gene panel developed in our group identified a novel ferroportin p.D84E variant in a patient with iron overload. Wild‐type and mutant ferroportin constructs were generated, transfected into HEK293 cells and analysed using an all‐in‐one flow‐cytometry‐based assay to study the effects on hepcidin‐mediated internalization and iron transport. Consistent with the classical phenotype of ferroportin disease, the p.D84E mutation results in an inability to transport iron and hepcidin insensitivity. These results validate a recently proposed 3D‐structural model of ferroportin and highlight the significance of this variant in the structure and function of ferroportin. Our novel ferroportin antibody and assay will be valuable tools for investigating the regulation of hepcidin/ferroportin function and the development of novel approaches for the therapeutic modulation of iron homeostasis.