It has been observed earlier that human blood group B galactosyltransferase (GTB) hydrolyzes its donor substrate UDP-Galactose (UDP-Gal) in the absence of acceptor substrate, and that this reaction is promoted by the presence of an acceptor substrate analog, α-L-Fuc-(1,2)-β-d-3-deoxy-Gal-O-octyl (3DD). This acceleration of enzymatic hydrolysis of UDP-Gal was traced back to an increased affinity of GTB toward the donor substrate in the presence of 3DD. Herein, we present new thermodynamic data from isothermal titration calorimetry (ITC) on the binding of donor and acceptor substrates and analogs to GTB. ITC data are supplemented by surface plasmon resonance and STD-NMR titration experiments. These new data validate mutual allosteric control of binding of donor and acceptor substrates to GTB. It is of note that ITC experiments reveal significant differences in enthalpic and entropic contributions to binding of the natural donor substrate UDP-Gal, when compared with its analog UDP-Glucose (UDP-Glc). This may reflect different degrees of ordering of an internal loop (amino acids 176–194) and the C-terminus (amino acids 346–354), which close the binding pocket on binding of UDP-Gal or UDP-Glc. As both ligands have rather similar dissociation constants KD and almost identical modes of binding this finding is unexpected. Another surprising finding is that an acceptor analog, α-L-Fuc-(1,2)-β-d-3-amino-3-deoxy-Gal-O-octyl (3AD) as well as the constituent monosaccharide β-d-3-amino-3-deoxy-Gal-O-octyl (3AM) effectively inhibit enzymatic hydrolysis of UDP-Gal. This is unexpected, too, because in analogy to the effects of 3DD one would have predicted acceleration of enzymatic hydrolysis of UDP-Gal. It is difficult to explain these observations based on structural data alone. Therefore, our results highlight that there is an urgent need of experimental studies into the dynamic properties of GTB. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 784–795, 2013.