Objective To observe the structure of human skin equivalent (HSE) model in vitro, and to preliminarily assess the barrier function of HSE. Methods The HSE model was established by two steps: preparation of dermal layer and preparation of full-thickness skin. Keratinocytes were inoculated on the surface of dermal layer prepared by type 1 collagen coating fibroblasts. After submerged cultured for one week, air-liquid interface cultured for three weeks to enhance the proliferation of keratinocytes, the HSE model was obtained. Then the macroscopical structure and electon microscopical structure of HSE were studied. To investigate the barrier function of HSE model, indometacin was selected as the model drug for the penetration testing in vitro. Results The structures similar to dermal layer and epidermal layer of human skin were developed in HSE model, furthermore, complete straum corneum with similar ultrastructure of human skin was developed on the surface of epidermal layer. The penetration testing showed that the barrier function of HSE was slightly lower than that of rat abdominal skin within 24 h, but the cumulative content (Q24) of indometacin of HSE was far slower (only 28%) than rat abdominal skin from 22 to 24 h. However, the steady state flux of indometacin showed no significant difference in HSE model and rat abdominal skin(P > 0.05). Conclusion The morphology and structure of HSE model were close to human skin; and in HSE model, the barrier function against indometacin was almost as the same as the rat abdominal skin. It is expected that HSE model be an alternative option of animal skin to evaluate the effect of transdermal drug delivery system. [ABSTRACT FROM AUTHOR]