Abstract: Objective To simplify the method for separation and cultivation of rat testicular Sertoli cells with high viability, quantity and expression efficiency. Methods Testicular Sertoli cells from 2 to 3-week-old male Wistar rats were prepared by digestion with collagenase, trypsin and DNase and cultured together with active lymphocytes to observe their killing effect against lymphocytes. After cell culture for 72 h, the Sertoli cells were morphologically observed by different means and identified with transmission electron microscope. Fas ligand and follicle-stimulating hormone receptor (FSHR) were examined immunohistochemically to identify testicular Sertoli cells. SABC method was used for labeling the Fas ligand on the testicular Sertoli cells. Results The viability of the isolated and cultured Sertoli cells was more than 90%, and in in vitro culture, Sertoli cells, which expressed the Fas ligand, could kill the active lymphocytes. Conclusion This method improves the efficiency in acquisition of rat testicular Sertoli cells expressing Fas ligand, which are believed to be a potential donor for co-transplantation with parathyroid cells to offer immune privilege.