Poly(3-(2-methoxyethyl)-N-vinyl-2-pyrrolidone) (MeOE-PVP), a potential material in the pharmaceutical industry, is an amphiphilic polymer with hydrophobic CH groups and two different hydrophilic groups (CO and C–O), and readily absorbs water vapor from humid environments. In this work, the hydration capabilities and structures of different chemical groups in MeOE-PVP film during water vapor sorption and desorption process are investigated by in situ FTIR spectroscopy, two-dimensional (2D) correlation analysis and density functional theory (DFT) calculations. When the film is tested in a stable humid environment, the changes of the CO group are more sensitive to water vapor than those of the C–O group, which is further verified by 2DIR maps, with much more hydrated structures of the CO group being formed after moisture absorption. Compared with MeOE-PVP in aqueous solution, it is believed that C–O group is a relatively stable hydrophilic group from which is difficult to lose water molecules once it is at the hydrated state. In addition, combined with DFT calculations, it is observed that CO and C–O groups of MeOE-PVP are inclined to interact with water molecules in the form of CO⋯H2O and CO⋯H2O⋯H2O, but as CO⋯(H2O)2, probably due to limited absorption concentration and contact area in the MeOE-PVP film.