Nonwoven nanofiber matrices were prepared by electrospinning a solution of silk fibroin (SF) dissolved either in formic acid or in 1,1,1,3,3,3-hexafluoro-2-isopropyl alcohol (HFIP). The mean diameter of the electrospun nanofibers prepared from SF dissolved in formic acid was 80 nm with a unimodal size distribution, which was smaller than those prepared from HFIP (380 nm). SF nanofibers were then treated with an aqueous methanol solution, and structural changes due to solvent-induced crystallization of SF were investigated using IR and 13C solid-state CP/MAS NMR spectroscopy. SF nanofibers prepared from formic acid were found to have a higher proportion of β-sheet conformations than those prepared from HFIP. Methanol treatment provided a fast and effective means to alter the secondary structure of both types of SF nanofibers from a random coil form to a β-sheet form. As demonstrated in the present study, this approach to controlling the dimensions and secondary structure of proteins using various solvents may be useful for the design and tailoring of materials for biomedical applications, especially for tissue engineering applications.