Abstract: The mechanical properties of silk fiber from regenerated silk fibroin (RSF) aqueous solution can be improved and greatly affected by posttreatment. In order to optimize the posttreatment process for the RSF fiber, the orientation and conformation development of the RSF molecules in two different posttreatment processes were studied quantitatively. The two processes are named as method A (the RSF fiber was immersed in φ=80% ethanol solution for 1 h then drawn 2 times in steam) and method B (the RSF fiber was drawn 2 times in φ=80% ethanol solution then immersed in the same solution for 1 h). Furthermore, the relationship between the conformation transition and the mechanical properties of the RSF fibers was discussed. Comparing to immersing the fiber in ethanol aqueous solution, it is more efficient to transit the RSF conformation and improve the RSF orientation by postdrawing the fiber in ethanol aqueous solution or in steam. Thus, method B instead of method A was adopted for an optimal process due to the resultant high βsheet content, orientation and excellent mechanical properties of the RSF fibers.