Abstract: Zein film was prepared under different zein concentration, glycerol ratio and forming temperature, and the change of their mechanical properties during 24 days storage at different relative humidity (RH)was determined. The results showed that tensile strength (TS)of the film prepared under different preparing conditions decreased firstly and then increased while the elongation at break (EB)changed on the contrary at RH34%, RH43%, and RH54% during 24 d storage. TS of the film prepared under different zein concentration and forming temperature increased gradually, and EB decreased rapidly at RH80%, but the trend of TS and EB of film with different glycerol ratio at RH80% was in accordance with those at RH34%, RH43%, and RH54%. The change of mechanical properties of zein film at RH65% was affected markedly by the forming condition. In general, effect of RH on the mechanical properties of zein film was more obvious than their preparing condition. Ambient humidity of film application should be taken into the effect factor in order to develop effective film.