d-tagatose, a rare sugar with broad applications in the food and pharmaceutical industries, can be synthesized from d-fructose by epimerases, an approach with significant biosynthesis potential. However, so far there has been only one attempt to engineer tagatose 4‑epimerase through random mutagenesis. The lack of exploration has severely restricted the application of epimerases in the industrial production of d-tagatose. The previous research primarily focused on enhancing the catalytic activity of epimerases through random mutagenesis, with limited understanding of how substrate-adjacent residues affect enzyme efficiency, which was hence the subject of this study. First, using a semi-rational design approach, after three rounds of mutagenesis and screening, the mutant (S125D/S268A/Q124E) of D-tagaturonate epimerase (UxaE) from Thermotoga neapolitana was obtained, which increased d-tagatose production efficiency by 10.8-fold compared to the wild type, with a specific activity of 1505.6 U/g, representing the highest specific activity reported for a UxaE mutant to date. Molecular dynamics simulations showed that the substitution of Ser to Asp-125 enhanced substrate-enzyme interactions, while the substitution of Glu to Gln-124 reinforced the hydrogen bond network around the catalytic residue Glu-128, further reshaping the substrate-binding pocket and enhancing catalytic activity. This study for the first time explored the enhancement of the C4 epimerization function of epimerases by reshaping their substrate-binding pockets, thereby providing a feasible route for the industrial biosynthesis of d-tagatose.