The regenerated Antheraea pernyi silk fibroin films were prepared from calcium nitrate solution and crystallized with aqueous methanol solution. The structural and conformational changes were investigated by the X-ray diffraction method and infrared spectroscopy upon methanol treatment. The concentration and treatment time of aqueous methanol solution have a great influence on the conformation of regenerated films. The conformational change from a random coil to beta-sheet structure was occurred within 5 min by the treatment of 40-60% methanol solution. As the methanol concentration increases up to 100%, the transition time of conformational change is delayed, and the structural change is not occurred in the case of 100% methanol used. The content of a alpha-helix, beta-sheet, and random coil conformation was calculated and examined to find out the effect of methanol treatment on the conformation changes. The beta-sheet structure can be transformed from a random coil, while the content of alpha-helix structure is not changed, regardless of methanol treatment.