This work describes for the first time the preparation of cross-linked and non-cross-linked silk fibroin (SF) microspheres using the simple water-in-oil emulsion solvent diffusion method. Aqueous SF solution and ethyl acetate were used as water and oil phases, respectively. Span80, oil-soluble emulsifier was found to induce the formation of SF microparticles that are completely spherical in shape and smooth in surface. SF microsphere sizes were found to depend upon various process parameters. The SF microsphere matrices showed predominantly random coil conformation. It was possible to fabricate genipin-cross-linked SF microspheres by cross-linking SF solution with genipin before microsphere formation. Both non-cross-linked and genipin-cross-linked SF microspheres contained porous structures. Percentage of dissolution in water decreased and density values of the SF microspheres increased when increasing the genipin ratio and cross-linking time. The genipin cross-linking induced SF conformational transition from random coil to beta-sheet form but the size and shape of the SF microparticles did not change. It is suggested that these SF microspheres might be suitable microcarriers for hydrophilic drug delivery. (C) 2010 Elsevier B.V. All rights reserved.