A combined approach based on the us ATR-FT/IR and steady-state fluorescence spectroscopy allowed to shed light on the effects of the additive methoxypolyethylene glycol (MePEG) on the hydration, conformtion and dynamic properties of lipase from Burkholderia cepacia dehydrated to form a film. Spectroscopic data show that the additive has little effect on the structure of the protein; however, H/D exchange kinetic and fluorescence anisotropy suggest a more flexible enzyme molc when in the presence of MePEG. By infrared spectroscopy, we estimated that, after conditioning the films at water activity of 1, the water content in the lipase dehydrated with MePEG is 5.4- and 4.7-fold higher than in absence of the additive and the additive alone, respectively. Additionally, our infrared data suggest that MePEG by hindering intermolecular protein-protein interact and contributing to increase the accessibility and flexibility of the lipase in the dehydrated solid film. These factors also explain the enhancement of the enzyme catalytic activity (i.e., up to 3.7-fold in neat organic solvent) when in the presence of MePEG. The method and results presented might better address the use of additives for preparation of enzymes employed in non-aqueous media or of proteins used in a dry form in different fields of biotechnology.