Epidermis reconstructed on de-epidermized dermis (RE-DED) and on fibroblast-populated collagen matrix (Living Skin Equivalent) showed a histologic resemblance to native epidermis. However, some abnormalities have been found including different expression pattern of differentiation markers from native epidermis. In this study, to reconstruct an epidermis model resembling native epidermis more closely than previous skin equivalents, de-epidermized dermis (DED) was raised on fibroblast-populated collagen matrix and keratinocytes were cultured on top of the DED at the air-liquid interface. The new skin equivalent like RE-DED showed a similar morphology to that of native epidermis. Immunohistochemical studies revealed that differentiation markers such as involucrin, loricrin and filaggrin but not keratin 1 expressed similar pattern characteristics to native epidermis compared with those of RE-DED. In addition, the new model showed some fibroblasts in the DED as a result of migration from the fibroblast-populated collagen matrix, mimicking a living dermis in vivo. These results indicate that the new model seems to be a better skin equivalent model than previous models. Also, they provide additional evidence that the presence of fibroblasts improves epidermal differentiation.