The mechanical denaturation process of silk fibroin is examined by Raman spectroscopy. The fresh silk fibroins from the middle gland of mature silkworms are drawn to various ratios on a tensile tester ( R = ldrawn/ linitial, where l is length) and their conformations are measured with Raman spectroscopy. Undrawn silk fibroin is mainly in the random coil structure with some α-helical conformation, the characteristic bands appearing at 1252 and 1660 (random coil) and at 942, 1106, and 1270 cm−1 (α-helix). When the samples are drawn up to R = 4 at an extension rate of 500 mm/min, two peaks at 1233 cm−1 (the amide III band) and 1085 cm−1 appear; it is shown that the β-sheet conformation is then formed. With an increase in drawing ratios, the intensities of these β-sheet bands increase and those of the random coil and α-helical bands decrease gradually. These changes indicate that, under the action of stress, the conformation of fibroin is altered from random coil and α-helix to β-sheet structures. This result is quite similar to the results achieved by the spinning of the silkworm. The effect of the water content in liquid silk on this conformational transition process is revealed and discussed.