β‐1,4‐galactosyltransferase 1 (β4gal‐T1, EC 2.4.1.38) transfers galactose from UDP‐galactose to free N‐acetyl‐d‐glucosamine or bound N‐acetyl‐d‐glucosamine‐R. Soluble β4gal‐T1, purified from human milk has been refractory to structural studies by X‐ray or NMR. In a previous study (Malissard et al. 1996, Eur. J. Biochem. 239, 340–348) we produced in the yeast Saccaromyces cerevisiae an N‐deglycosylated form of soluble β4gal‐T1 that was much more homogeneous than the human enzyme, as it displayed only two isoforms when analysed by IEF as compared to 13 isoforms for the native β4gal‐T1. The propensity of recombinant β4gal‐T1 to aggregate at concentrations > 1 mg·mL⁻¹ prevented structural and biophysical studies. In an attempt to produce a β4gal‐T1 form suitable for structural studies, we combined site‐directed mutagenesis and heterologous expression in Escherichia coli. We produced a mutated form of the catalytic domain of β4gal‐T1 (sfβ4gal‐T1mut) in which seven mutations were introduced at nonconserved sites (A155E, N160K, M163T, A168T, T242N, N255D and A259T). Sfβ4gal‐T1mut was shown to be much more soluble than β4gal‐T1 expressed in S. cerevisiae (8.5 mg·mL⁻¹ vs. 1 mg·mL⁻¹). Catalytic activity and kinetic parameters of sfβ4gal‐T1mut produced in E. coli were shown not to differ to any significant extent from those of the native enzyme.