Chitosan membrane was prepared in various conditions and diffusive permeabilities of theophylline and vitamin B-12 were investigated. The membrane preparation procedure consists of dissolving chitosan in acid solution, cast on the glass plate, drying the dope solution, and immersion of the plate in the gelating agent. Effects of the kinds of acids to dissolve chitosan, chitosan concentration, drying time of the dope solution, and the kinds of the gelating agent on the membrane structure and performance were studied in detail. With increasing the chitosan concentration, the solute permeability decreased, while the selectivity of theophylline to vitamin B-12 increased. The membrane changed from the wholly porous structure to the asymmetric structure by the increase of the chitosan concentration. Furthermore, the use of ethanol as the gelating agent brought about the wholly porous structure with the high permeability and the low selectivity. The asymmetric structure and the wholly dense structure were obtained in the cases of the gelating agents, such as aqueous NaOH solution and dimethyl sulfoxide (DMSO), respectively. Thus, the membrane structure can be controlled by the kinds of gelating agent.