Sporothrix schenckii and Sporothrix brasiliensis are causative agents of sporotrichosis, a mycosis that affects humans and other mammals. Adhesins are considered among the primary virulence factors of these species; however, their molecular identities are currently scarce. Here, we generated silenced mutant strains in PAP1, encoding a peptidorhamnomannan-associated protein, in both fungal species and compared their phenotypical characterization. PAP1-silenced mutants had normal growth, dimorphism, and morphology, suggesting the gene is not essential. The silenced strains showed defects in adhesion to HeLa cells, laminin, fibronectin, fibrinogen, type-I collagen, and type-II collagen. The S. schenckii silenced mutants showed a significant reduction in the adhesion to elastin, whilst the S. brasiliensis mutant showed impaired ability to bind thrombospondin 1 and to form biofilms. Both sets of PAP1-silenced mutants displayed defects in the cell wall composition, with reduced levels of cell wall rhamnose. The S. schenckii mutants showed a compensatory mechanism increasing cell wall mannose content, while the S. brasiliensis mutants increased cell wall protein levels in compensation to PAP1 silencing. Both mutant sets changed their ability to stimulate cytokine production in human peripheral blood mononuclear cells and monocyte-derived macrophages. In addition, phagocytosis of S. schenckii PAP1-silenced strains was significantly increased, while the S. brasiliensis PAP1-silenced strains were poorly phagocytosed. Finally, PAP1 silencing affected virulence in both species. These results suggest that S. schenckii and S. brasiliensis PAP1 code for a protein with adhesive properties to different ligands. This is a cell wall protein that contributes to Sporothrix virulence and the interaction with immune cells.