The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to b sheet and the b sheet aggregation growth of silk fibroin are identified in the B. mori regenerated silk fibroin aqueous solution by circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in prion protein, amyloid b (Ab) protein, and a-synuclein protein with the conformation transition from a soluble protein to a neurotoxic, insoluble b sheet containing aggregate, is a novel suggestion for the silk spinning process. We present evidence that two steps are involved in this mechanism: (a) nucleation, a ratelimiting step involving the conversion of the soluble random coil to insoluble b sheet and subsequently a series of thermodynamically unfavorable association of b sheet unit, i. e. the formation of a nucleus or seed; (b) once the nucleus forms, further growth of the b sheet unit becomes thermodynamically favorable, resulting a rapid extension of b sheet aggregation. The aggregation growth follows a first order kinetic process with respect to the random coil fibroin concentration. The increase of temperature accelerates the b sheet aggregation growth if the b sheet seed is introduced into the random coil fibroin solution. This work enhances our understanding of the natural silk spinning process in vivo.