We have determined the functions of the enzymes encoded by the lnpB, InpC, and InpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine I-phosphate; the InpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine I-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDPgalactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.