Histoplasma capsulatum is a dimorphic fungal pathogen that survives and replicates within macrophages (MΦ). Studies in human and murine MΦ demonstrate that the intracellular growth of H. capsulatum yeasts is exquisitely sensitive to the availability of iron. As H. capsulatum produces hydroxamate siderophores, we sought to determine if siderophores were required for intracellular survival in MΦ, and in a murine model of pulmonary histoplasmosis. The expression of SID1 (coding for L-ornithine-N(5)-monooxygenase) was silenced by RNA interference (RNAi) in H. capsulatum strain G217B, and abolished by gene targeting in strain G186AR. G217B SID1-silenced yeasts grew normally in rich medium, did not synthesize siderophores, and were unable to grow on apotransferrin-chelated medium. Their intracellular growth in human and murine MΦ was significantly decreased compared to wild type (WT) yeasts, but growth was restored to WT levels by the addition of exogenous iron, or restoration of SID1 expression. Similar results were obtained with G186AR Δsid1 yeasts. Compared to WT yeasts, G217B SID1-silenced yeasts demonstrated in C57BL/6 mice significantly reduced growth in the lungs and spleens seven days after infection, and 40% of the mice given a normally lethal inoculum of G217B SID1-silenced yeasts survived. These experiments demonstrate that: (1) SID1 expression is required for siderophore biosynthesis by H. capsulatum strain G217B, (2) SID1 expression is required for optimum intracellular growth in MΦ, and (3) inhibition of SID1 expression in vivo reduces the virulence of H. capsulatum yeasts.