Objective: To investigate the effect of co-transplantation of islet and mesenchymal stem cells (MSCs) to induce immune tolerance on islet graft, and to compare the efficacies of MSCs via different delivery routes. Methods: SD and Lewis rats were used as donor and recipient, respectively. MSCs were isolated from the femur of the SD rats with adherence culture method and then expanded in vitro. Islets were also isolated from the donor pancreas by collagenase-V digestion. Forty recipient Lewis rats with streptozotocin induced diabetes were equally randomized into group A (islets combined with BrdU-labeled MSCs were transplanted via portal vein), group B (islets were transplanted through portal vein and BrdU-labeled MSC were infused via tail vein), group C (islets transplantation through portal vein combined with cyclosporine administration) and group D (islets transplanted via portal vein without any other intervention). Blood glucose levels of the recipient rats were observed after transplantation and the survival times of islet grafts in each group were compared. On the 7th day after transplantation, the samples of liver, thymus gland and spleen were taken from some of the living donors to observe homing sites of MSCs. Results: Both group A and B had the longest maintenance times of normal blood glucose level, followed by group C, and that of group D was the shortest. The survival time of islet graft in group A was (12.1±2.3) d, group B was (8.6±1.4) d, group C was (13.2±1.9) d and group D was (2.2±0.6) d, respectively. MSCs homing observation showed that in group A, the BrdU-positive MSCs were mainly located in the liver, and some of them formed the “microencapsulation” that enclosed islet grafts, while MSCs were mainly observed in the thymus gland and spleen in group B. Conclusion: Co-transplantation of islets and MSCs can promote immune tolerance for islet grafts, and MSCs delivery through portal vein has better efficacy than that observed through peripheral vein.