Regenerated Antheraea pernyi silk fibroin films prepared from calcium nitrate solution were treated with heat for crystallization. The structural and conformational changes caused by heat treatment were investigated with X-ray diffraction, infrared spectroscopy, and differential scanning calorimetry. The temperature and treatment time of heat greatly influenced the conformation of the regenerated films. The conformational change was caused by heat treatment over 230 degreesC, and the transition was enhanced by increasing time and temperature. The contents of alpha -helix, P-sheet, and random-coil conformations were calculated and examined to investigate the effect of heat treatment on the conformational changes. The beta -sheet structure could be transformed from a random-coil conformation on heat treatment, whereas the content of ct-helix structure was nearly unchanged, regardless of heat-treatment conditions.