In this work, the nature of physisorbed water and its impacts on the structure, surface chemistry, and proton conduction properties of TiO2 nanocrystals were investigated by a combinational spectral technique. All TiO2 nanocrystals were directly prepared by a hydrothermal method, which showed highly hydrated and sulfated surfaces. The surface water molecules were indicated to exist in a wide set of energetically nonequivalent surface hydration groups, leading to the removal of physisorbed and chemisorbed water in sequence with increasing temperature. After heating treatment at 100 ℃ in air, physisorbed water layers were recovered with no significant impacts on the TiO2 nanostructure. On the other hand, when treated at the same temperature in vacuum, the recovery of physisorbed water layers was partially reversible, while a new hydration state appeared due to the filling of the high-energy adsorption sites by water molecules, which led to a significant increase in the amount of water molecules for surface hydration and an accelerated dehydration process toward lower temperature. As a result, an abnormal increase was observed in proton conductivity. These observations were explained in terms of thermally induced changes of surface chemistry and the amount of hydrated water. The results reported in this work are important, which may help understand the roles that the physisorbed water plays in stabilizing the nanostructures and therefore could have a broad class of implications.