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The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) by the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), catalyzed by the β-galactosidase from Bacillus circulans (BcβGal), was studied in hydro-organic media, starting from o-nitrophenyl-β-D-galactopyranoside (oNPG) as a galactosyl donor. Thermal stability and synthesis activity of BcβGal were shown to depend on the organic solvent polarity, characterized by its Log P value. BcβGal was thus most stable in 10% (v/v) t-BuOH, an organic solvent found to have a stabilizing and/or weakly denaturing property, which was confirmed for high t-BuOH concentrations. In the same manner, the optimal synthesis yield increased as the Log P value of the organic solvent increased. The best results were obtained for reactions carried out in 10% (v/v) pyridine or 2-methyl-2-butanol, which gave 47% GlcNAc transgalactosylation yield based on starting oNPG, of which 23% (11 mM; 4.3 g/L) consisted in LacNAc synthesis. Furthermore, it was also established that both the GlcNAc transgalactosylation yield and the enzyme regioselectivity depended on the percentage of organic solvent used, the optimal percentage varying from 10 to 40% (v/v), depending on the solvent. This phenomenon was found to correlate mainly with the thermodynamic activity of water (a(w)) in the aqueous organic solvent mixture, which was found to be optimal when close to 0.96, whatever the organic solvent used. Finally, this study highlighted the fact that the regioselectivity of BcβGal for 1-4 linkage formation could be advantageously managed by controlling the a(w) parameter.