The 13C CP/MAS NMR, X-ray diffraction, and Raman spectroscopies were used for monitoring the structural transition of Samia cynthia ricini (S. c. ricini) silk fibroin induced by stretching. Here the silk fibroin was obtained from the aqueous solution stored in the silk gland. All of these spectroscopic data indicate that the structural transition from R-helix to â-sheet occurs with increasing the stretching ratio, especially between the stretching ratios, 4 and 6. The 13C chemical shifts of Ala Câ peak in the 13C CP/MAS NMR spectrum change significantly depending on R-helix, random coil, and two kinds of â-sheet structure, which make it possible to clarify the local structure and structural transition. Actually, the fraction of individual structure in the silk fibroin samples was determined from decomposition of the Ala Câ peak by assuming a Gaussian line shape. To examine the conformational change of Ala residues of the polyalanine region in S. c. ricini silk fibroin observed with these spectroscopic methods, MD simulations for four peptide molecules, AGGAGG(A)12GGAGAG, with R-helix conformation were performed in the presence of water molecules under different tensile strengths, and then additional MM calculations were performed after removal of water molecules. The change in the conformational character of Ala residues induced by stretching is explicable by these calculations.