BACKGROUND Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dermatophytosis caused by T. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin ®, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection. METHODS The reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation. RESULTS The histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of T. rubrum infection in an intuitionistic way. CONCLUSIONS We established the T. rubrum infection model on RHE in vitro successfully. It is a promising model for further investigation of the mechanisms involved in T. rubrum infection.