Introduction: Recognition of fungal surface β-glucan by pattern recognition receptor Dectin-1 is a critical process for fungal clearance in the lung. In humans, persistent fungal infection is observed in individuals with particular Dectin-1 polymorphism. We have identified that nitric oxide (NO) modifies critical cysteines in pattern recognition molecules to disassemble and alter protein function. There is a hydrophobic S-nitrosylation motif present in surfactant protein-D (SP-D) that is also present in Dectin-1. We hypothesized that Dectin-1 can be modified by nitrosative stress potentially leading to impairment of fungal clearance. Materials and Methods: Recombinant Dectin-1 was incubated with l-nitrosocysteine (L-SNOC) and S-nitrosylated Dectin-1 was detected by Biotin-switch assay. Cells of a murine macrophage line (Raw 264.7) were incubated with S-nitroso-glutathione (GSNO) and Dectin-1 shedding from the cell surface was determined by Western blot. Dectin-1 quaternary structure was determined by native gel electrophoresis. Dectin-1 function was assayed by NF-κB activity and IL-6 mRNA real-time polymerase chain reaction (PCR). Phagocytic activity was measured by fluorescence labeled zymosan beads. Results: Dectin-1 was S-nitrosylated by l-nitrosocysteine (L-SNOC) in vitro, as determined by Biotin-switch assay, resulting in structural disruption. We used Western blotting and flow cytometry to demonstrate that incubation of a murine macrophage cell line (Raw 264.7 cells) with GSNO reduced the surface Dectin-1 expression as a result of shedding to the media. The shedding of Dectin-1 is due to formation of S-nitrosothiol (SNO)-Dectin-1 and disruption of the Dectin-1 oligomeric complex. GSNO also induces Dectin-1 shedding from the cell surface. The functional significance of GSNO treatment of macrophages is shown by reduced β-glucan-mediated signaling in terms of NF-κB function and IL-6 expression. Finally, it was demonstrated that GSNO treatment reduces the capability of macrophages to phagocytose zymosan. Conclusions: These data provide mechanistic data to support the role of Dectin-1 nitrosylation as a mediator of reduced fungal clearance in the face of increased NO exposure.