The transglycosylation activity of a novel commercial β-galactosidase from Bifidobacterium bifidum (Saphera) was evaluated. The optimal conditions of operation of this enzyme, measured with o-nitrophenyl-β-D-galactopyranoside, were 40 °C and pH around 6.0. Although at low lactose concentrations the character of this enzyme was basically hydrolytic, an increase of lactose concentration to 400 g/L resulted in a significant formation (107.2 g/L, 27% yield) of prebiotic galactooligosaccharides (GOS).The maximum amount of GOS was obtained at a lactose conversion of approximately 90%, which contrasts with other β-galactosidases, for which the highest GOS yield is achieved at 40-50% lactose conversion. Using HPAEC-PAD, semipreparative HPLC-HILIC, MS, 1D and 2D NMR, we determined the structure of most of the GOS synthesized by this enzyme.The main identified products were Gal-β(13)-Gal-β(14)-Glc (3´-O-Beta-galactosyllactose), Gal-β(1→6)-Glc (allolactose), Gal-β(13)-Glc (3-galactosyl-glucose), Galβ(1→3)-Gal (3-galactobiose) and the tetrasaccharide Gal-β(13)-Gal-β(13)-Galβ(14)-Glc. In general, the B. bifidum β-galactosidase showed a tendency to form β(13) linkages followed by β(16), and more scarcely β(14).