: Iron is an essential element for almost all organisms. In eukaryotes, it is mainly used in mitochondria for the biosynthesis of iron-sulphur clusters and heme-group maturation. Iron is delivered into the mitochondrion by mitoferrins, members of the mitochondrial carrier family (MCF), through an unknown mechanism. In this article, the yeast homologs of these proteins, Mrs3p and Mrs4p, were studied by inserting them into liposomes. In this context, they could transport iron (II) across the proteoliposomes membrane, as revealed using the iron-chelator bathophenanthroline. A series of amino acid-modifying reagents were screened for their effects on Mrs3p-mediated iron transport. Results suggested that carboxylic and imidazole groups are essential for iron transport. This was confirmed by in vivo complementation assays, which demonstrated that three highly conserved His residues are important for Mrs3p function. These His residues are not conserved in other MCF members, thus they are likely to play a specific role in iron transport. A model describing how these residues help iron to transit smoothly across the carrier cavity is proposed and compared to structural and biochemical data available for other carriers in this family.