The macrophage is the primary host cell for the fungal pathogen Histoplasma capsulatum during mammalian infections, yet little is known about fungal genes required for intracellular replication in the host. Since the ability to scavenge iron from the host is important for the virulence of most pathogens, we investigated the role of iron acquisition in H. capsulatum pathogenesis. H. capsulatum acquires iron through the action of ferric reductases and the production of siderophores, but the genes responsible for these activities and their role in virulence have not been determined. We identified a discrete set of co-regulated genes whose transcription is induced under low iron conditions. These genes all appeared to be involved in the synthesis, secretion, and utilization of siderophores. Surprisingly, the majority of these transcriptionally co-regulated genes were found clustered adjacent to each other in the genome of the three sequenced strains of H. capsulatum, suggesting that their proximity might foster coordinate gene regulation. Additionally, we identified a consensus sequence in the promoters of all of these genes that may contribute to iron-regulated gene expression. The gene set included L-ornithine monooxygenase (SID1), the enzyme that catalyzes the first committed step in siderophore production in other fungi. Disruption of SID1 by allelic replacement resulted in poor growth under low iron conditions, as well as a loss of siderophore production. Strains deficient in SID1 showed a significant growth defect in murine bone-marrow-derived macrophages and attenuation in the mouse model of infection. These data indicated that H. capsulatum utilizes siderophores in addition to other iron acquisition mechanisms for optimal growth during infection.