Fructose is not only an important food and beverage ingredients, but also a renewable resource for production of 5-hydroxymethylfurfural. This study investigated nontoxic basic amino acids (arginine, lysine, and histidine) as isomerization catalysts to isomerize glucose to fructose in water. The results showed that arginine was the most effective isomerization catalyst with a 31% maximum fructose yield with 76% selectivity achieved under the investigated reaction conditions. A mechanistic study verified that the isomerization reaction catalyzed by arginine proceeded through an enediol intermediate, which formed after the deprotonation at C-2 position of acyclic glucose.