Recent studies have suggested that adipose tissue-derived mesenchymal stem cell (ADSC) therapy and OX40 costimulation blockade are two immunomodulatory strategies used to suppress the immune response to alloantigens. However, relatively little has been reported regarding the immunomodulatory potential of the abilityof these two strategies to synergize. Thus, in the present study, we aimed to investigate OX40-Ig fusion protein (OX40Ig) expression in ADSCs and to validate their more potent immunosuppressive activity in preventing renal allograft rejection. For this purpose, ADSCs from Lewis rats were transfected with the recombinant plasmid, pcDNA3.1(-)OX40Ig, by nucleofection. The ADSCs transduced with the plasmid (termed ADSCsOX40Ig) or untransduced ADSCs (termed ADSCsnative) were added to allostimulated mixed lymphocyte reaction (MLR) in vitro. In vivo, ADSCsOX40Ig, ADSCsnative, or PBS were administered to an allogeneic renal transplantation model, and the therapeutic effects, as well as the underlying mechanisms were examined. The results revealed that both the ADSCsnative and ADSCsOX40Ig significantly suppressed T cell proliferation and increased the percentage of CD4+CD25+ regulatory T cells in allogeneic MLR assays, with the ADSCsOX40Ig being more effective. Furthermore, the results from our in vivo experiments revealed that compared with the ADSCsnative or PBS group, the administration of autologous ADSCsOX40Ig markedly prolonged the mean survival time of renal grafts, reduced allograft rejection, and significantly downregulated the mRNA expression of intragraft interferon-γ (IFN-γ) , and upregulated the mRNA expression of interleukin (IL)‑10, transforming growth factor-β (TGF-β) and forkhead box protein 3 (Foxp3). The findings of our study indicate that the use of ADSCsOX40Ig is a promising strategy for preventing renal allograft rejection. This strategy provides the synergistic benefits of ADSC immune modulation and OX40-OX40L pathway blockade, and may therefore have therapeutic potential in clinical renal transplantation.