Regenerated cellulose-based films combined with glycerol and polyvinyl alcohol (PVOH) show interesting UV-light barrier properties, with potential application in food packaging to prevent oxidative deterioration. However, these materials are sensitive to moisture, and their properties could be modified as a function of the relative humidity. Hence, the objective of the present work was to evaluate the changes in the main properties of regenerated cellulose-glycerol-PVOH films depending on the relative humidity. Using the GAB adsorption isotherms, the moisture content was related with the water activity of the films at several relative humidity conditions. According to the obtained results, water molecules manifested a plasticizing effect modifying the mechanical, water vapour permeability and optical properties of the developed films. Tensile strength and Young's modulus values ranged from 92.65 to 17.57 MPa and from 3639.09 to 227.89 MPa, respectively. Both of them decreased when the moisture content increased. The mechanical resistance to deformation of films enhanced at high moisture content, changing from 5.88 to 15.97% and from 0.59 to 2.97 mm in the tensile and puncture test, respectively. This effect was also observed for the burst strength. Water vapour permeability increased from 5.15·10−10 to 5.44·10−9 g/ms Pa when the moisture content increased, being more significative at higher values. No significant variations were observed in the UV-VIS transmittance at different moisture contents. The obtained results allow expanding the knowledge of the behavior of films based on regenerated cellulose.