Objective To determine the effects of CD25siRNA transfection in corneal graft on the transplantation rejection in rats undergoing penetrating keratoplasty, and on the expression of IL-10 and FOXP3 in the graft. Methods Fifty μL EntransterTM-control CD25siRNA and 50 μL EntransterTM-CD25siRNA were formulated as nanoparticle transfection reagents, and 50 μL normal saline was defined as control. Orthotopic corneal transplantation was performed only on the right eyes of Wistar SD rats. Corneal recipients were divided into the control group, EntransterTM-control CD25siRNA group and EntransterTM-CD25siRNA group, with 28 rats in each group. After surgery, the recipient eyes were examined daily for 21 d, and HE staining, immunohistochemistry, Western blotting and qRT-PCR were performed on 3, 7, 14 and 21 d postoperatively. Results The survival curves indicated that CD25siRNA treatment could significantly prolong graft survival time compared to the other 2 groups (P<0.05). HE staining showed that CD25siRNA treatment could alleviate infiltration of inflammation cells and reduce neovascularization. Meanwhile, lower CD25 expression and higher IL-10 expression were detected in the EntransterTM-CD25siRNA group compared with the other 2 groups (P<0.05), and FOXP3 expression exhibited no significantly difference among the groups (P>0.05). Conclusion CD25siRNA transfection significantly prolongs the median survival time of corneal allografts and increases the graft survival rate via up-regulating anti-inflammatory molecule IL-10 and inhibiting tissue inflammatory response.