Pancreatic islet transplantation is a promising treatment for type 1 diabetes (T1D). Interleukin-35 (IL-35) is a recently discovered cytokine that exhibits potent immunosuppressive functions. However, the role of IL-35 in islet transplant rejection remains to be elucidated. In this study, we isolated islet cells of BALB/c mouse and purified CD4+ T cell subsets of a C57BL/6 mouse. The model for islet transplantation was established in vitro by co-culture of the islet cells and CD4+ T cells. IL-35 (20 ng/ml) was administered every other day. Following co-culture, the islet function and Treg/Th17 ratio were analyzed on days 1, 3, and 5. Furthermore, the Th17/Treg ratio was modulated (1:0-2), and the function of islet cells as well as proliferation of Th17 cells were analyzed. T cell sorting was performed using the magnetic bead sorting method; Treg and Th17 count using flow cytometry; cell proliferation detection using the carboxyfluorescein diacetate succinimidyl ester (CFSE) method, and islet function test using the sugar stimulation test. Results showed that Th17 counts increased in the co-culture system. However, after administration of IL-35, the number of Treg cells increased significantly compared to that in the control group (50.7% of total CD4+ T cells on day 5 in IL-35 group vs. 9.5% in control group) whereas the proliferation rate of Th17 cells was significantly inhibited (0.3% in IL-35 group vs. 7.2% in control group on day 5). Reducing the Th17/Treg ratio significantly improved the function of transplanted islets. Treg inhibited Th17 proliferation and IL-35 enhanced this inhibitory effect. IL-35 mitigates the function of murine transplanted islet cells via regulation of the Treg/Th17 ratio. This might serve as a potential therapeutic strategy for in-vivo islet transplant rejection and T1D.