• The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule-specific Vacuolar iron Transporter-Like (VTL) proteins in Medicago truncatula. • Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA-seq data showing differential expression of VTL4 and VTL8 during early and late infection, respectively. • The vtl4 insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly-arranged VTL4–VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed in vtl4 mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. • These data indicate that VTL8, the closest homologue of SEN1 in Lotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids.