CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe2+ into protoporphyrin IX. Here, we find that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 μM. Circular dichroism spectra of CyaY suggested that binding of heme to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.