Homologs of the β-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Galβ1→3GalNAc, GNB) than that on lacto-N-biose I (Galβ1→3GlcNAc). K m value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k cat value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB. © 2009 Springer-Verlag.