ABSTRACT: Biodegradable titanium dioxide (TiO2)/whey protein isolate (WPI) blend films were made by casting denatured WPI film solutions incorporated with TiO2 nanoparticles. X-ray diffraction, UV-vis spectra, and fluorescence spectra of the films showed the successful incorporation of TiO2 nanoparticles into the WPI matrix and indicated the interactions between TiO2 and WPI. Mechanical tests revealed the antiplasticizing effect of TiO2 nanoparticles on the WPI/TiO2 film. Small amounts (<1 wt%) of added TiO2 nanoparticles significantly increase the tensile properties of WPI film, but also decrease the moisture barrier properties. The addition of higher amounts (>1 wt%) of TiO2 improves moisture barrier properties but lowers the tensile properties of the film. Microstructural evaluation confirmed the aggregation and distribution of TiO2 nanoparticles within the WPI matrix and validated the results of functional properties of the WPI/TiO2 film.