Room temperature ionic liquids (ILs) can affect enzyme activity in some enzyme-catalyzed reactions, however the effects of these cosolvents on the enzymes are not clearly understood. Using β-galactosidase from Thermus thermophilus HB27 (TTP0042), we found an important change from the classical regioselectivity of the transglycosylation reaction with this enzyme. The enzyme increases N-acetyl-D-lactosamine synthesis (Galβ[1→4]GlcNAc) when RTILs are used instead of the traditional self-condensated products. To understand the possible effect of these liquids on the synthetic behavior of the enzyme, we performed a molecular interaction study by surface plasmon resonance. The KD value obtained for this interaction could mean that ILs bind to β-galactosidase through non specific interactions characterized by very fast kinetics and millimolar affinity. Then, several reactions were performed, increasing the concentration of the IL. As a result, a dependence on the IL's concentration was found for transglycosylation products. We hypothesize that ILs might induce conformational changes in the enzyme, which would modify the enzymatic activity and regioselectivity. These structural modifications were confirmed in the secondary and tertiary structures of the protein by circular dichroism and fluorescence studies, respectively. Molecular modeling confirms this hypothesis and shows that the enzyme becomes more flexible in an IL–water mixture and that it allows stabilization of the GlcNAc molecule in the active centre of the enzyme, in order to develop a new product according to the original regioselectivity of the reaction.