Background: The cornified cell envelope (CE) which is formed during the terminal differentiation of keratinocytes, is a specialized structure which forms a structurally and functionally complete permeability barrier. Objective: The purpose of our study is to investigate the effects of changes in the calcium ions on keratinocyte differentiation, especially in the expression of CE protein. Methods: The permeability barrier of hairless mice was disrupted by tape-stripping and then exposed to the air or occluded with a water-vapor impermeable membrane, and iontophoresis was done without permeability barrier perturbation. Skin specimens were prepared for ion capture cytochemistry and immunohistochemistry with anti-K5, anti-K10, anti-K6, anti-involucrin and anti-loricrin. Results: The calcium gradient which disappeared after tape-stripping was restored at 36 h after tape-stripping with air exposure and at 60 h after tape-stripping with occlusion. The change in calcium ions produced by both positive and negative iontophoresis showed recovery at 6 h. Expression of basal K5 showed a slight decrease and expression of suprabasal K10 showed an increase at 12 h with air exposure after tape-stripping, tape stripping with occlusion, and iontophoresis. Expression of K6 appeared at 12 h after tape-stripping and then in the whole epidermis at 36 h with air exposure after tape-stripping and tape stripping with occlusion and focally appeared in the stratum granulosum and stratum spinosum after iontophoresis. Expression of involucrin was increased at 12 h with air exposure after tape-stripping and iontophoresis and was extended to the lower spinous layers in tape-stripping with occlusion. Expression of loricrin in air exposure after tape-stripping, tape-stripping with occlusion and iontophoresis was similar to that of normal skin. Conclusion: The changes in calcium ions without permeability barrier perturbation are related to the expression of CE protein. It is thought that calcium ions in the epidermis have an important role in the terminal differentiation of keratinocytes.