A combination of ethanol solvent and hydrotalcite (HT) with a Mg/Al ratio of 3:1 was effective for the isomerization of glucose to fructose in up to 56% yield with a high selectivity of 80%. The ethanol solvent shifts the isomerization equilibrium between glucose and fructose, which allows the fructose yield to exceed the upper limit of enzymatic isomerization conducted in water at modest temperatures (i.e., 50%). The HT catalyst maintained such high catalytic performance in repeated use at least three times via calcination and subsequent reconstruction of the original layered structure by the memory effect, which was operated in an aqueous solution containing ammonium carbonate. The results of catalyst screening, characterization, and isotope tracer analysis have revealed that the base sites of HT mainly contribute to its high catalytic activity for glucose-to-fructose isomerization.