A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.