An anisotropic proton conductive polymer film was prepared from poly(decylacrylamide-co-12-acrylamidododecanoic acid). The polymer film was prepared on a solid substrate by spin coating and the structure was analyzed by X-ray diffraction measurement. The initial polymer film exhibited featureless diffraction, which indicates that the film was amorphous. On the other hand, when the film was annealed at 60°C under 98% RH, the film structure was converted from amorphous to highly oriented lamellar structure. The proton conductivities of the film parallel and perpendicular to the substrate surface were studied by impedance measurement. The conductivity at the parallel direction increase whereas that at the perpendicular direction decrease with measurement time. This is because the film formed a lamellar structure in-situ during the proton conductivity measurement. In other words, the anisotropic conduction state can be reached as long as the proton conductivity measurements were carried out.