Silk fibroin (SF) microspheres were prepared by the simple water-in-oil emulsion solvent diffusion method without any surfactants. Aqueous SF solution and dichloromethane were used as water and oil phases, respectively. Influence of water: oil phase ratios on SF microsphere characteristics was investigated. From FTIR spectra, the resulting SF microspheres showed predominantly random coil SF conformation. SF microspheres observed from SEM images were spherical with deflated surfaces in some cases. Particle sizes of the SF microspheres were in the range 45-92 μm. Finally, bovine serum albumin (BSA) was used as a model protein for entrapment within the SF microspheres. The BSA-loaded SF microspheres were larger than unloaded SF microspheres. In vitro release tests indicate that BSA release from the SF microspheres was influenced by BSA content. © 2010 Korean Institute of Chemical Engineers, Seoul, Korea.