Interleukin‐35 (IL‐35) is a cytokine recently discovered to play a potent immunosuppressive role by intensifying the functions of regulatory T cells and inhibiting the proliferation and functions of T helper 1 and T helper 17 cells. Mesenchymal stem cells (MSCs) have recently emerged as promising candidates for cell‐based immune therapy, and our previous study showed that IL‐35 gene modification can effectively enhance the therapeutic effect of MSCs in vitro. In this study, we isolated adipose tissue‐derived MSCs in vitro and infected them with lentiviral vectors overexpressing the IL‐35 gene, thereby creating IL‐35‐MSCs. Subsequently, IL‐35‐MSCs were then injected into mice of the allogeneic heterotopic abdominal heart transplant model to determine their effect on allograft rejection. The results showed that IL‐35‐MSCs could continuously secrete IL‐35 in vivo and in vitro, successfully alleviate allograft rejection and prolong graft survival. In addition, compared to MSCs, IL‐35‐MSCs showed a stronger immunosuppressive ability and further reduced the percentage of Th17 cells, increased the proportion of CD4+ Foxp3+ T cells, and regulated Th1/Th2 balance in heart transplant mice. These findings suggest that IL‐35‐MSCs have more advantages than MSCs in inhibiting graft rejection and may thus provide a new approach for inducing immune tolerance during transplantation.