The non-biodegradable and non-renewable nature of plastic packaging has led to a renewed interest in packaging materials based on bio-nanocomposites (biopolymer matrix reinforced with nanoparticles such as layered silicates). Bio-nanocomposite films based on soy protein isolate (SPI) and montmorillonite (MMT) were prepared using melt extrusion. Effects of the pH of film forming solution, MMT content, and extrusion processing parameters (screw speed and barrel temperature distribution) on the structure and properties of SPI-MMT bio-nanocomposite films were investigated. X-ray diffraction (XRD), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were used for structural characterization of the films. Properties of the films were determined by tensile testing, dynamic mechanical analysis (DMA), thermogravimetric analysis (TGA), and water vapor barrier measurement. The arrangement of MMT in the soy protein matrix ranged from exfoliated at lower MMT content (5%) to intercalated at higher MMT content (15%). There was a significant improvement in mechanical (tensile strength and percent elongation at break) and dynamic mechanical properties (glass transition temperature and storage modulus), thermal stability, and water vapor permeability of the films with the addition of MMT. The results presented in this study show the feasibility of using bio-nanocomposite technology to improve the properties of biopolymer films based on SPI. (C) 2010 Elsevier Ltd. All rights reserved.