Dectin-1 is a C-type lectin receptor that recognizes fungal β-glucan, and mediates the production of reactive oxygen species and inflammatory cytokines. Thus Dectin-1 is thought to be essential for anti-fungal immune responses. Murine Dectin-1 mRNA is alternatively spliced and generates two isoforms (isoform A and B). Human Dectin-1 mRNA is also alternatively spliced and its functional isoforms (isoform A and B) are structurally similar to each of the mouse isoforms. One of the major differences among the four murine and human isoforms is the position and number of N-linked glycosylation motifs. But the significance of the glycosylation to the recognition of β-glucan is not known. In this paper, using various glycosylation consensus sequence mutants, we demonstrated that the N-linked glycosylation of Dectin-1 affects the cell surface expression of the molecule. The expression levels on the cell surface influence the ligand-binding and the collaboration with TLR2 in the activation of NF-κB. These results suggest that N-linked glycosylation on Dectin-1 is essential for the recognition of fungal β-glucan and subsequent activation of NF-κB.