We examined the sugar-cleaving abilities of β-galactosidases from jack bean and Streptococcus towards sugars containing fucose residues, and found that jack bean β-galactosidase has an ability to cleave the β1-3 linkage between galactose (Gal) and fucose (Fuc) residues, but not β1-4 linkage. On the other hand, streptococcal β-galactosidase was found to cleave the linkage in both Galβ1-4Fuc and Galβ1-3Fuc disaccharide units. Such a difference in sugar-cleaving abilities between these 2 β-galactosidases will be useful for structural analysis of glycans, especially those from species belonging to Protostomia, such as Caenorhabditis elegans.