Ferroportin (Fpn)-the only known cellular iron exporter-transports dietary and recycled iron into the blood plasma, and transfers iron across the placenta. Despite its central role in iron metabolism, our molecular understanding of Fpn-mediated iron efflux remains incomplete. Here, we report that Ca²⁺ is required for human Fpn transport activity. Whereas iron efflux is stimulated by extracellular Ca²⁺ in the physiological range, Ca²⁺ is not transported. We determine the crystal structure of a Ca²⁺-bound BbFpn, a prokaryotic orthologue, and find that Ca²⁺ is a cofactor that facilitates a conformational change critical to the transport cycle. We also identify a substrate pocket accommodating a divalent transition metal complexed with a chelator. These findings support a model of iron export by Fpn and suggest a link between plasma calcium and iron homeostasis.