Abstract： Objective To establish the method for isolation and culture of porcine Sertoli cell sand detect the expression of the immune privilege-related molecules in the cultured cells. MethodsTestes were aseptically removed from the 10-to 15-days old Large White piglets. Testes were decap-sulated, minced and then digested with 0.2% (W/V) collagenase type V, 0.25% (W/V) trypsin and 0.05% (W/V) DNaseI. In the primary isolation, Sertoli cells were cultured at 37℃with 5% CO2.Inverted phase contrast microscopy and HE staining were used to observe the morphology of Sertoliceils, and the Sertoli cells were identified under an electron microscope. Viability and apoptosis ofcultured cells were measured with the AnnexinV-PI staining by flow cytometry. The expression of sox9, FasL, TGF-β and clusterin in Sertoli cells was detected by RT-PCR. The viability of long-termcultured Sertoli cells was assayed by MTT. Results In the cultured total cells, Sertoli cells accountedfor more than 90%. The apoptosis rate and mortality of Sertoli cells was (2.61±0.96)% and (2.12±0.74)% respectively. RT-PCR revealed that the expression of sox9, TGF-β and clusterin in theSertoli cells was strongly positive, but FasL was weakly positive. Viability of cultured cells measuredby MTT conformed that the sertloli cells could survive more than 21 days. Conclusion The isolationand culture methods of the neonatal porcine Sertoli cells was established. Under the culturedconditions, the Sertoli cells can express the immune privilege molecules and successfully survive at least 21 days.