The separation of water/alcohol mixtures through chitosan membrane neutralized by various acids was investigated. The permselectivity of the neutralized chitosan membrane was affected by the species of acids and, only when neutralized by polybasic acids, was a marked increase of the selectivity observed and a high selectivity maintained for ion free mixtures. The marked increase of the selectivity could be explained by the contraction of the “holes” produced by the thermal motion of polymer chains due to the conformation change of chitosan molecule caused by the neutralization. The conformation change was confirmed by the analysis of the solid state structure of the neutralized chitosan using CP/MAS 13C-NMR.