Structure of Bombyx mori silk fibroin membrane insolubilized with methanol, especially in the swollen state, is studied by means of spin-label ESR, 13C-NMR, and FT-IR (ATR method) spectroscopies. FT-IR data indicate that the conformational transition from random coil to antiparallel β-sheet occurs at the surface of the membrane upon immersion into 80% aqueous methanol. High resolution 13C-NMR observation of the membrane swollen in water shows that, the random coil portion whose segmental motion is very fast remains in the inner part of the swollen membrane. The fraction of this portion decreases with increasing methanol treatment time in the sample preparation. The heterogeneous structure of the swollen membrane was clarified from the complicated ESR spectra of the spin-labeled silk fibroin membranes. The ESR spectra were analyzed quantitatively and the fractions of the fast, slow, and very slow motions of the spin-label site (tyrosine side chain) were determined. A model is proposed for the heterogeneous structure of the swollen silk fibroin membrane.