An endo-β-1,3-glucanase from Agaricus brasiliensis ATCC 76739 was first produced and purified in the submerged culture. Purified endo-β-1,3-glucanase with a 13.1-fold purification and 6.7% yield was prepared by two steps: precipitation with 70% saturation ammonium sulfate and chromatography on hydrophobic interaction chromatography. The molecular mass of the enzyme was estimated to be 33 kDa by SDS-PAGE. The presence of endo-β-1,3-glucanase might explain the fall of the yield of bioactive exopolysaccharides in the late stage of the submerged cultures. The pH optimum for the enzyme was 4.5, and the temperature optimum was 45°C. The enzyme showed high pH stability within the range of pH 3.5-6.0 and thermostability up to 50°C, and exhibited a half-life of 30 min at 55°C, which was better than that of a thermophilic fungus, Scytalidium thermophilum. The enzyme activity was strongly inhibited by HgCl2.