Polymer vesicles with intrinsic molecular permeability have attracted attention as structural components for nano/microreactors in the context of drug delivery and artificial cells. However, the molecular mechanism of the permeability has not yet been elucidated. Herein, we report the relationship between the hydration in the hydrophobic region of molecular permeable polymer vesicles and the molecular permeability. Neutron scattering measurements revealed that the hydrophobic layer is highly hydrated and that the degree of hydration increases upon lowering the solution temperature. Most notably, fluorescence-bleaching-after-recovery experiments showed that the permeability of the vesicles does not increase with increasing solution temperature and that the highest permeability was observed in the coldest solution. These results suggest that increasing the degree of hydration in the hydrophobic layer represents a major factor in enhancing the permeability. This study can thus be expected to provide guidelines for the design of polymer vesicles with intrinsic molecular permeability.