Abstract: Silk Fibroin (SF) microparticles containing hollow structure were prepared by a water-in-oil emulsion solvent diffusion method without any surfactants. Aqueous SF solution and ethyl acetate were used as water and oil phases, respectively. Influences of SF concentration and post methanol treatment on microparticle characteristics were investigated. All microparticles contained open hollow structures. Microparticle sizes increased with the SF concentration. Conformation of SF microparticles determined from FTIR spectra changed from predominantly random coil to β-sheet form after methanol treatment. Particle surfaces of methanol-treated SF microparticles were rougher than those of non-treated. The SF concentrations and methanol treatment did not affect open hollow structure of the SF microparticles. Major advantages of this method are the elimination of surfactants and extractable pore templates. These hollow SF microparticles are expected to be potential used as biodegradable microcarriers of cell and protein drug, because their hollow structures should permit cell attachment and enzyme immobilization.