Objective: To set up a method for isolation and culture of mature Sertoli cells and to estimate their effects on allogeneic islets cocultured in vitro. Methods: Adult SD rat testicular Sertoli cells were prepared successfully by three-step enzyme digestion. Then they were cocultured respectively with allogeneic islets and activated Wistar rat splenocytes. 24-hour cumulative insulin release and glucose-stimulated insulin secretion test were performed to detect islet function between pure islets culture group and coculture group. Splenocyte proliferation activity was determined by MTT colorimetry assay to observe the inhibition effect of Sertoli cells in different densities. Result: Firstly, in pure islet culture group, the 24-hour cumulative insulin release was gradually decreased in 21-day culture time. Compared to day 3, this change was significant on day 7 (P ＜ 0.05) and on day 10,14,21 (P ＜ 0.01). In contrast, in coculture group, compared to day 3, the 24-hour cumulative insulin release was increased significantly on day 7 (P ＜ 0.01 ), and then gradually decreased on day 10 and 14, but still higher than that of day 3. It was on day 21 that it began to decrease compared to day 3 (P ＜ 0.05). During the culture time in vitro, the 24-hour cumulative insulin release of islet coculture group was significantly higher than that of pure islets culture group (P ＜ 0.01). In the case of stimulation index(SI), there was a similar tendency as insulin release in the two groups. Secondly, mature Sertoli cells(1×106/mL)pretreated by 15 grays irradiation could decrease proliferation activity of activated splenocytes compared to that of control group (P ＜ 0.01 ). This inhibition effect was dose-dependent. Conclusion: Mature Sertoli cells can improve the function and prolong the survival of islet cells cultured in vitro. They can also provide an immune protection to islet cells. The approach described above might be applicable to human islet transplantation as soon as if it is also valid in large animal models.