Self-sustained populations of captive red panda were challenged by low reproductive success rate. Defining the different types of testicular cell and increasing the knowledge of male fertility was critical for the improvement of reproductive efficiency in red panda captive populations. Here, we firstly report the histological structure in red panda testes at various ages. Besides, our data also showed that markers for red panda testicular cells including ITGA6, PLZF, THY1, VASA, DAZL, GATA4, VASA, and αSMA were expressed in adult testis tissue. To further examine the localizations of ITGA6, VASA, VIM, and αSMA, the immunohistochemical analysis was performed and the result showed the typical staining patterns of undifferentiated spermatogonia, multiple types of germ cells, Sertoli cells, and peritubular myoid cells (PMCs), respectively. For the testicular tissue digestion, two-step enzyme digestion was used to obtain the cell suspension. The red panda Sertoli cells, expressing Sertoli cell marker VIM, were cultured and conserved in this study, which can be used for red panda Sertoli cell establishment and as the feeder cells for red panda spermatogonial stem cells (SSCs) growing.