We previously reported that Fusarium oxysporum 12S produces two bifunctional proteins, FoAP1 and FoAP2, with alpha-D-galactopyranosidase (GPase) and beta-L-arabinopyranosidase (APase) activities. The aim of this paper was to purify a third GPase, FoGP1, from culture supernatant of F. oxysporum 12S, to characterize it, and to determine its mode of action towards gum arabic. A cDNA encoding FoGP1 was cloned and the protein was overexpressed in Escherichia coli. Module sequence analysis revealed the presence of a GH27 domain in FoGP1. The recombinant enzyme (rFoGP1) showed a GPase/APase activity ratio of 330, which was quite different from that of FoAP1 (1.7) and FoAP2 (0.2). Among the natural substrates tested, rFoGP1 showed the highest activity towards gum arabic. In contrast to other well-characterized GPases, rFoGP1 released a small amount of galactose from a-galactosyl oligosaccharides such as raffinose and exhibited no activity toward galactomannans, which are highly substituted with a-galactosyl side chains. This indicated that FoGP1 is an unusual type of GPase. rFoGP1 released 30% of the total galactose from gum arabic, suggesting the existence of a large number of agalactosyl residues at the non-reducing ends of gum arabic side chains. Together, rFoGP1 and alpha-L-arabinofuranosidase released four times more arabinose than alpha-L-arabinofuranosidase acting alone. This suggested that a large number of alpha-L-arabinofuranosyl residues is capped by a-galactosyl residues. H-1 NMR experiments revealed that rFoGP1 hydrolyzed the alpha-1,3-galactosidic linkage within the side chain structure of [alpha-D-Galp-(1 -> 3)-alpha-L-Araf-(1 ->] in gum arabic. In conclusion, rFoGP1 is highly active toward alpha-1,3-galactosyl linkages but negligibly or not active toward alpha-1,6-galactosyl linkages. The novel FoGP1 might be used to modify the physical properties of gum arabic, which is an industrially important polysaccharide used as an emulsion stabilizer and coating agent.