l-Galactose (l-Gal) is one of the components of plant cell wall polysaccharides. In the GDP-l-fucose-deficient Arabidopsis thaliana mutant mur1, l-fucose (l-Fuc) residues in xyloglucan are substituted by l-Gal residues. l-Gal only differs from l-Fuc by the presence of an oxygen at C-6. Thus, we hypothesized that the A. thaliana xyloglucan α1,2-l-fucosyltransferase (AtFUT1) is also responsible for the l-galactosyl transfer to d-galactose residues in xyloglucan. In this study, we heterologously produced AtFUT1 in fission yeast and carried out an in vitro assay for the activities of AtFUT1 on GDP-l-Gal and xyloglucan oligosaccharide. We show that the recombinant AtFUT1 catalyzes l-Gal transfer to xyloglucan oligosaccharides although the initial velocity of l-Gal transfer is 3.1 times lower than that of l-Fuc transfer.