In this study, we investigated the localization, morphological features and cellular interactions of telocytes in the rat testicular interstitium. Transmission electron microscopy (TEM) and immunohistochemical and immunofluorescence analyses of the rat testicular interstitium showed a distinct layer of telocytes surround the seminiferous tubules along with inner layer of peritubular myoid cells. The majority of the telocytes were made up of a small cell body and moniliform prolongations that contained mitochondria and secretory vesicles. Some other telocytes were observed possessing large cell bodies. Within the testicular interstitium, the telocytes formed a network connecting peritubular myoid cells, Leydig cells as well as blood vessels. Immunohistochemical and double immunofluorescence analyses showed that rat testicular telocytes express CD34 and PDGFRα, but are negative for vimentin and α-SMA. Our findings demonstrate the presence of telocytes in the rat testicular interstitium. These cells interact with peritubular myoid cells, seminiferous tubules, Leydig cells and blood vessels via long telopode extensions, which suggests their vital role in the intercellular communication between different cell types within the rat testis.