In the testis of the Sunda porcupine (Hystrix javanica), the distribution pattern of cytoskeletal proteins was immunohistochemically examined to understand their functional roles in the testis. Immunoreactivity for α-smooth muscle actin (SMA) was found in the peritubular myoid cells of the seminiferous tubules and in the sub-tubular myoid cells of epididymal ducts. In intermediate filaments, desmin was exhibited in the sub-tubular myoid cells of the epididymal ducts, and vimentin immunoreactivity was detected in the Sertoli cells, Leydig cells, peritubular myoid cells, and sub-tubular myoid cells of the epididymal ducts. However, cytokeratin was not expressed in the testes or epididymides. Strong immunoreactivity for vimentin was demonstrated between the infranuclear part of the elevated nuclei and the basal membrane in the Sertoli cells. Furthermore, vimentin was extended from supranuclear to apical parts in some Sertoli cells. Immunoreactivity for α-tubulin was detected only in the Sertoli cells. It is concluded that, in the porcupine testes, desmin plays no role in the contractile function of peritubular myoid cells, vimentin may contribute to the anchoring and transition of the elevated nuclei in the Sertoli cells, and α-tubulin and vimentin in the Sertoli cells are likely involved in their structure and function as the main cytoskeleton.