The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an alpha-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that alpha-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked alpha-D-Glcp residues substituted at position 6 with alpha-D-Glcp branches. Soluble alpha-glucan, but not beta-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when alpha-glucan from conidial surface was removed by enzymatic treatment with alpha-amyloglucosidase, thus indicating an essential role of alpha-glucan in P. boydii internalization by macrophages. alpha-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with alpha-amyloglucosidase. Finally, alpha-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and alpha-glucan could be a target for intervention during fungal infections.