The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1(+)-otg3(+) (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2(+) gene product had Gal transfer activity toward a pyridylaminated Man(9)GlcNAc(2) oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3(+) gene product exhibited Gal transfer activity toward the pyridylaminated Man(9)GlcNAc(2) oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, (1)H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides.