Silk fibroin from Bombyx mori is a high-molecular-weight protein, largely employed in the biomaterials field. Several parameters can affect the folding and assembly of fibroin heavy and light chains. The present work has shown that anisotropic and water-stable films are produced when fibroin solution is cast under an alternating electric field (AC). The treatment can affect the mechanical, thermal and surface properties of fibroin films. These effects have been related to the alignment of molecular dipoles and the formation of oriented supramolecular assemblies. Cell response is affected by this novel processing: MRC5 fibroblasts, cultured on anisotropic fibroin films, preferentially spread parallel to the field direction 6 h after seeding. [Figure: see text].