Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1-3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1-3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223 and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein-protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling.