Objective: The aim of the current study was to unravel the underlying mechanisms of interleukin-35 (IL-35) in inducing immune tolerance in renal transplantation rats by modulating CD4+CD25+ regulatory T-cells. Methods: From a total of 60 Sprague-Dawley (SD) rats, 40 were selected, randomly, for preparation of allograft renal transplantation. They were divided into two groups, the donor group and recipient group. The remaining rats were assigned to the control group. Test group rats were further assigned to test groups A and B, while the control group rats were assigned to control groups A and B. Intravenous infusion of IL-35 was used as the intervention for rats in group A. Rats in group B received intravenous infusion of normal saline. Eight days later, the rats were sacrificed and peripheral blood samples were collected for measurement of serum levels of creatinine (Cr) and blood urea nitrogen (BUN). Serum levels of IL-10,1L-17, and IL-23 were detected using enzyme-linked immunosorbent assays. Percentages of CD4+CD35+ regulatory T-cells were measured using flow cytometry. Moreover, mRNA expression of Foxp3 in renal tissues was determined by polymerase chain reaction. Results: In the experimental group and control group A, significant (P<0.05) decreases were observed in levels of Cr, BUN, IL-10, IL-17, and IL-23, compared with levels in the experimental group and control group B, respectively. Of the four groups, the highest levels of Cr, BUN, IL-10, IL-17, and IL-23 were found in rats of test group B. The lowest levels were found in control group A. There were no significant (P>0.05) differences between the two control groups concerning ratios of CD4+CD25+ regulatory T-cells. However, the ratio in test group A was significantly (P<0.05) higher than that in test group B. Furthermore, expression levels of Foxp3 in test group A and control group A were significantly (P<0.05) higher than expression levels in test group B and control group B, respectively. Foxp3 expression in test group B was significantly (P<0.05) higher than that in control group A. The highest expression of Foxp3 was recorded in test group A, while the lowest expression was recorded in control group B. Conclusion: IL-35 can affect renal function in renal transplantation rats, as well as expression of IL-6, IL-17, and IL-23. Underlying mechanisms involve IL-35 regulating CD4+CD25+ regulatory T-cells to induce immune tolerance by targeting Foxp3 in renal transplantation rats.