Histoplasma capsulatum is a dimorphic fungus that causes respiratory and systemic disease and is capable of surviving and replicating within macrophages. The virulence of Histoplasma has been linked to cell wall α-(1,3)-glucan; however, the role of this polysaccharide during infection, its organization within the cell wall, and its synthesis and regulation remain poorly understood. To identify genes involved in the biosynthesis of α-(1,3)-glucan, we employed a forward genetics strategy to isolate physically marked mutants with reduced α-(1,3)-glucan. Insertional mutants were generated in a virulent strain of H. capsulatum by optimization of Agrobacterium tumefaciens-mediated transformation. Approximately 90% of these mutants possessed single insertions with no chromosomal rearrangements or deletions in the host genome. To confirm the role and specificity of identified candidate genes, we phenocopied the disrupted locus by either RNA interference or targeted gene deletion. Our findings indicate α-(1,3)-glucan production requires the function of the AMY1 gene product, a novel protein with homology to the α-amylase family of glycosyl hydrolases, and UGP1, a UTP-glucose-1-phosphate uridylyltransferase which synthesizes UDP-glucose monomers. Loss of AMY1 function attenuated the ability of Histoplasma to kill macrophages and to colonize murine lungs.