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The isomerization of D-glucose to D-fructose plays a key role in the biochemical and chemical conversion of biomass, and it is therefore desirable to develop and improve catalysts for this reaction. In this study, the environmentally friendly polymer poly(amidoamine) (PAMAM) dendrimer's properties as catalysts for this isomerization are investigated. The experimental results showed that the PAMAM dendrimers, which have basic terminal groups, can effectively promote the D-glucose isomerization reaction. Under the optimized reaction conditions, D-fructose was generated with a 20% maximum yield and above 90% selectivity. 13C and 2 H isotope experiments by NMR were carried out to explore the reaction mechanism. When the reaction was performed in D 2 O, the C1 signal of D-fructose changed to a triplet, which confirmed that the C1 carbon binds to a deuterium atom, i.e., isotopic exchange. It was also found that the deuterium atom at the C2 position of D-glucose-2-d 1 cannot transfer to D-fructose. These data indicate that PAMAM dendrimers catalyze D-glucose isomerization through a mechanism, which includes deprotonation, formation of ene-diol intermediate, and proton exchange with the solvent.