A series of chitosan membranes and their N-acetylated counterparts were prepared and their differences in acidic solution stability and thermal and mechanical properties were studied. The differences were attributed to differences in crystallinity of the membrane as the result of preparation conditions and N-acetylation. The results show that after N-acetylation, the acidic solution stability of N-acetylated membranes increased and the tensile strengths and the enthalpies of those membranes prepared from lower DD chitosans became higher and those from higher DD ones became lower. However, the swelling index of those modified membranes prepared from lower DD chitosans decreased and that from higher DD chitosans increased. Original membranes were prepared by using chitosans with different degrees of deacetylation (DD) and solution pHs to manipulate different chain flexibilities.