In response to iron starvation, Staphylococcus aureus secretes both staphyloferrin A and staphyloferrin B, which are high-affinity iron-chelating molecules. The structures of both HtsA and SirA, the ferric-staphyloferrin A [Fe(III)-SA] and ferric-staphyloferrin B [Fe(III)-SB] receptors, respectively, have recently been determined. The structure of HtsA identifies a novel form of ligand entrapment composed of many positively charged residues. Through ionic interactions, the binding pocket appears highly adapted for the binding of the highly anionic siderophore SA. However, biological validation of the importance of the nine SA-interacting residues (six arginines, one tyrosine, one histidine, and one lysine) has not been previously performed. Here, we mutated each of the Fe(III)-SA-interacting residues in HtsA and found that substitutions R104A, R126A, H209A, R306A, and R306K resulted in a reduction of binding affinity of HtsA for Fe(III)-SA. While mutation of almost all proposed ligand-interacting residues decreased the ability of S. aureus cells to transport (55)Fe(III)-SA, S. aureus expressing HtsA R104A, R126A, R306A, and R306K showed the greatest transport defects and were incapable of growth in iron-restricted growth media in a SA-dependent manner. These three residues cluster together and, relative to other residues in the binding pocket, move very little between the apo and closed holo structures. Their essentiality for receptor function, together with structural information, suggests that they form a positively charged platform that is required for initial contact with the terminal carboxyl groups of the two citrates in the Fe(III)-SA complex. This is a likely mechanism by which HtsA discerns iron-bound SA from iron-free SA.