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Understanding the mode of transport of water vapor through the film is important for improving the moisture barrier properties of wheat gluten (WG) films. Effective permeability (Peff), solubility (Seff), and diffusion (Deff) coefficients of a hydrophilic cast WG film were determined at 25°C within the relative humidity (RH) range of 0–84% (with a 9–13% RH gradient between upstream and downstream water vapor flux). Peff, Seff, and Deff increased substantially as the RH gradient moved upwards in the RH spectrum. Peff increased by four orders of magnitude from the lowest RH condition of 0–11% (3.8×10−11 g·m/m2·s·Pa) to the highest RH condition of 75–84% (4.1×10−7 g·m/m2·s·Pa). A moisture sorption isotherm of the film at 25°C was constructed. Both the Guggenheim–Anderson–DeBoer (GAB) and the Kuhn moisture sorption isotherm models showed a good fit to the experimental adsorption data. Testing of WG films at the expected conditions of actual use is necessary to quantify the water vapor permeation through the films.