In the present study, preparation of silk fibroin (SF) microspheres with controllable conformational transition by water-vapor treatment for drug delivery was investigated. The SF microspheres with and without drug encapsulation were prepared by a water-in-oil emulsion solvent diffusion method. Average sizes of drug-free and drug-loaded SF microspheres were 158 and 167 mu m, respectively, as measured by light scattering analysis. They were spherical shape and smooth surface with high drug loading efficiency (83%). The water-vapor treatment with 90% relative humidity at ambient temperature induced SF conformational transition from random coil to -sheet form. Dissolution of the SF microspheres decreased as the treatment time increased. In vitro release of blue dextran model drug from the SF microspheres depends on the treatment time. It is considered that these water-vapor-treated SF microspheres have the potential to be developed further as drug delivery systems.