Iron is an essential cofactor for symbiotic nitrogen fixation. It is required by many of the enzymes facilitating the conversion of N2 into NH4+ by endosymbiotic bacteria living within root nodule cells, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Model legume Medicago truncatula Ferroportin2 (MtFPN2) is a nodule-specific gene that encodes an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature, and in the symbiosome membranes that contain the nitrogen-fixing bacteria in the differentiation and early-fixation zones of the nodules. Loss-of-function of MtFPN2 leads to altered iron distribution and speciation in nodules, which causes a reduction in nitrogenase activity and in biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that MtFPN2-facilitated iron delivery across symbiosomes is essential for symbiotic nitrogen fixation, while its presence in the vasculature does not seem to play a major role in in the conditions tested.