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In this study, we prepared regenerated silk fibroin (RSF) aqueous solution to approach the environmental condition in the gland of silkworm, Bombyx mori. Then electrospinning technique was used to prepare the silk fibers. The results showed that pH and concentration had a remarkable influence on the properties of RSF aqueous solutions. With the increase in concentration and the decrease in pH, the rheological behavior of RSF aqueous solutions exhibited a transition from Newtonion fluid to non-Newtonion fluid. At the same time, lowering the pH could induce gel formation and decrease the electrospinnable concentration of RSF aqueous solutions. With the decrease in pH and concentration, the morphology of the electrospun silk fibers changed from belt-like shape to uniform cylinder. The conformation of the electrospun silk fibers was characterized by RS, WAXD and DSC. It was found that electrospun fibers were predominantly random coil/silk I conformation.