Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter， ferroportin (Fpn) and a multicopper ferroxidase， that is， hephaestin (Heph)， ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However， whether APP family members interact with Fpn is unclear. Here， using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP， APLP1， and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured Fe efflux. We demonstrate that Fpn and Heph co-localize， and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast， none of the full-length， cellular APP proteins exhibited Fpn co-localization or FRET. Moreover， iron supplementation increased surface expression of the iron-efflux complex， and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization， addition of soluble E2 elements derived from APP and APLP2， but not APLP1， increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence， (K/R)EWEE， present in APP and APLP2， but not APLP1， helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.