β-1,3-Glucan constitutes a prominent cell wall component being responsible for rigidity and strength of the cell wall structure in filamentous fungi. Glycoside hydrolase (GH) family 81 endo-β-1,3-glucanases which can cleave the long chain of β-1,3-glucans play a major role in fungal cell wall remodeling. Here, we reported the complex structures of a fungal GH family 81 endo-β-1,3-glucanase from Rhizomucor miehei (RmLam81A), revealing the triple-helical β-glucan recognition and hydrolysis patterns. In the crystals, three structured oligosaccharide ligands simultaneously interact with one enzyme molecular via seven glucose residues, and the spatial arrangement of ligands to RmLam81A was almost identical to that of β-1,3-glucan triple-helical structure. RmLam81A performed an inverting catalysis mechanism with Asp475 and Glu557 severing as the general acid and base catalyst, respectively. Furthermore, two hydrophobic patches involving Tyr93, Tyr106, Ile108, Phe619 and Tyr628 alongside the ligand-binding site possibly formed parts of the binding site. A ligand-binding motif, β31-β32, consisting of two key residues (Lys622 and Asp624), involved the recognition of a triple-helical β-glucan. Our results provided a structural basis for the unique β-1,3-glucan recognition pattern and catalytic mechanism of fungal GH family 81 endo-β-1,3-glucanases, which may be helpful in further understanding the diverse physiological functions of β-1,3-glucanases.