The double-site variant (C450S-N475K) l-arabinose isomerase (l-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of d-galactose to d-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to d-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for d-galactose isomerization. The k (cat)/K (m) of the triple-site variant enzyme for d-galactose was 2.1-fold higher than for l-arabinose, whereas the k (cat)/K (m) of the double-site variant enzyme for l-arabinose was 43.9-fold higher than for d-galactose. These results suggest that the triple-site variant enzyme is a d-galactose isomerase. The conversion rate of d-galactose to d-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of l-arabinose to l-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for d-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for d-galactose and l-arabinose, and the triple-site variant enzyme has the potential to produce d-tagatose on an industrial scale.