The objective of the present study is to investigate the possibility of preparing pure protein microspheres from regenerated silk fibroin (RSF). It is found that RSF microspheres, with predictable and controllable sizes ranging from 0.2 to 1.5 µm, can be prepared mild self-assembling of silk fibroin molecular chains. The merits of this novel method include a rather simple production apparatus and no potentially toxic agents, such as surfactants, initiators, cross-linking agents, The results show that the particle size and size distribution of RSF microspheres are greatly affected by the amount of ethanol additive, the freezing temperature and the concentration of silk fibroin. Finally, the mechanism of RSF microspheres formation is also discussed based on our experimental results.