Abstract: The reaction mechanism of the isomerization of glucose to fructose and further dehydration of fructose to 5-hydroxymethylfurfural (5-HMF) in subcritical water was investigated by the dispersion-corrected density functional theory (DFT-D) method with Dmol³ package in Materials Studio. The implicit solvent model was used to evaluate the bulk solvation under the conductor-like screening model (COSMO) approach, in which a dielectric constant ( ε ) of 27 was used to represent the subcritical water at 523.15 K. The explicit solvent model was adopted with a hybrid micro-solvation-continuum approach, to indicate the micro-solvation by explicit H₂O molecules and the bulk solvation with ε = 27. The calculation results indicate that explicit H₂O molecules participate in the reaction and catalytically promote the proton transfer processes, suggesting that the explicit solvent model is preferable to the implicit solvent model to represent the conversion of 5-HMF in subcritical water. The isomerization of glucose to fructose is exothermic by 5.26 kcal/mol, where the isomerization of open-chain glucose to enol form is the rate-determining step, with the activation energy of 33.89 kcal/mol; the free energy of transition state configuration depends upon both the difficulty in α–H extraction of open-chain glucose and the stability of formed carbocation. In contrast, the hydration of fructose to 5-HMF is exothermic by 12.93 kcal/mol and the first hydration is the rate-determining step, with the activation energy of 50.59 kcal/mol; the free energy of transition state configuration is determined by the stability of carbocation formed by the dehydration of protonated OH group at C(2) site of fructose. This work discloses the promoting effect of Brønsted base on the isomerization of glucose to fructose and that of Brønsted acid on the dehydration of fructose to 5-HMF, which may provide certain clues to the modification of catalytic sites and the selection of solvent in the conversion of glucose to 5-HMF.