Regenerated silk fibroin (RSF) was prepared by dissolving in a CaCl2/ ethanol/H2O solvent system, freezing, and lyophilization. The effect of freezing temperature, alcohol addition, and molecular weight on the morphological and conformational changes were investigated through scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and differential scanning calorimetry analysis. However, the addition of a small amount of methanol induced the morphological change of RSF to a fine-particle aggregate, which resulted from the formation of a p-sheet crystalline structure. The lower the freezing temperature was, the more the formation of aggregates was favored, and the finer powder aggregates were formed. As the amount of added hydrophilic alcohol such as methanol and ethanol increased in the silk fibroin solution, a spherical powder form was changed to fine aggregates with the enhancement of thermal stability and crystallinity. On the other hand, RSFs prepared with a hydrophobic alcohol such as l-butanol or l-octanol showed a lump-like or sheet-like shape of morphology without any changes in conformational transition. It is concluded that the molecular weight of the silk fibroin and the type and amount of alcohol were determining factors in the morphological features of RSF, especially the size and shape of fibroin particles. A uniform ultrafine powder of RSF with a spherical form (similar to 1 mum) can be obtained when the molecular weight and the alcohol addition to the silk fibroin solution are controlled.