Intestinal iron absorption involves proteins located in the brush border membrane (BBM), cytoplasm, and basolateral membrane (BLM) of duodenal enterocytes. Ferroportin 1 (FPN1) and Hephaestin (Heph) are necessary for transport of iron out of enterocytes, but it is not known if these two proteins interact during iron absorption. We first examined co-localization of the proteins by cotransfection of HEK293 cells with pDsRed-FPN1 with pEmGFP-Heph or with the C-terminal truncated pEmGFP-Heph43 or -Heph685 and found that FPN1 and Heph with or without the C-terminus colocalized. In rat duodenal enterocytes, within 1 h of iron feeding prominent migration of FPN1 from the apical sub-terminal zone to the basal subnuclear zone of the BLM occurred and increased to at least 4 h after feeding. Heph exhibited a similar though less prominent migration after iron ingestion. Analysis using rat duodenal epithelial cell sheets demonstrated that: 1) by velocity sedimentation ultracentrifugation, FPN1 and Heph occupied vesicles of different sizes prior to iron feeding and migrated to similar fractions 1 hr after iron feeding; 2) by blue native (BN)/SDS PAGE, FPN1 and Heph interacted to form two complexes, one containing dimeric FPN1 and intact Heph and the other consisting of monomeric FPN1 and a Heph fragment; and 3) by immunoprecipitation, anti-Heph or anti-FPN1 antiserum co-immunoprecipitated FPN1 and Heph. Thus, the data indicate that FPN1 and Heph migrate and interact during iron feeding and suggest that dimeric FPN1 is associated with intact Heph.