Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of ␤-1,3-galactan main chains and ␤-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-␤-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed ␤-1,3-galactan, but did not act on ␤-1,3-glucan, ␤-1,3:1,4-glucan, xyloglucan, and agarose. It released various ␤-1,3-galactooligosaccharides together with Gal from ␤-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on ␤-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-␤-1,3-glucanase and endo-␤-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-␤-1,3-galactanase.