Different sterilization techniques such as Co(60) gamma irradiation and autoclaving were used to treat the regenerated Bombyx mori silk fibroin (RSF) in aqueous solutions. The effect of the two above mentioned sterilization methods on the conformational changes and gelation rate of RSF was studied. According to the analysis of circular dichroism spectroscopy, gamma irradiation may initiate the conformational transition from random coil to β-sheet for RSF, while autoclaving shows less significant influence on the structure changes of RSF in aqueous solutions. The results also indicate that gelation time decreased to 5 days after γ-irradiation treatment while gelation time increased to 31 days after autoclave treatment. Moreover, particles of RSF in solutions changed larger and gelation time increased when autoclaving treatment time extended. However, no detectable changes of RSF secondary structure were found investigated by XRD and FTIR. Which indicated that under both sterilization methods, the RSF structure was transformed from random coil to β-sheet structure after gelation.