The BLLJ_1391 protein from Bifidobacterium longum subsp. longum JCM1217, a cytosolic β- N -acetylglucosaminidase belonging to glycoside hydrolase family (GH) 20, hydrolyzed lacto- N -triose II (LNTri) as well as chitin oligosaccharides. Its reaction was found to follow a substrate-assisted mechanism with anomeric retention, which is common for GH 20 enzymes. Homologous enzymes are found in genomic sequences of B. longum subsp. infantis , Bifidobacterium bifidum , and Bifidobacteium breve , all of which are infant gut-associated species of Bifidobacterium . The distribution resembles that of 1,3-β-galactosyl- N -acetylhexosamine phosphorylase, suggesting that the enzyme plays a role in metabolism of human milk oligosaccharides by hydrolyzing LNTri generated via the cytosolic hydrolysis of lacto- N -tetraose (LNT) by LNT 1,3-β-galactosidase.