The relationship between the preparation conditions and the function of silk fibroin membrane was investigated in terms of water-sorption, membrane potential, ion permeability, X-ray diffraction and birefringence. We clarified the influences of preparation conditions of the silk fibroin membrane such as the concentration of fibroin solution (dilute and concentrated), substrate (acrylic resin and mercury) and drying rate (slow and rapid), on membrane potential and ion permeability of the fibroin membrane. The orientation function of the amorphous region is remarkably dependent on the preparation conditions, whereas that of crystal region is almost independent. When the mercury was used as a substrate and the drying rate is slow, the aggregation state of the fibroin membrane estimated from the iso-electric point, IEP was the highest. The membrane prepared from a concentrated solution on mercury at the slow-drying rate has the smallest average pore radius _??_. On the other hand, those for the membranes prepared from a dilute solution on an acrylic sheet are generally larger. It was clarified that the change of ion permeability of the membrane generally agreed to the order of IEP and _??_ of the membrane.