Bone marrow-derived mesenchymal stem cells (BMSCs) are promising gene vehicles for cancer gene therapy. In our previous study, we reported that BMSCs expressing interleukin (IL)-18 effectively inhibit the growth of glioma in rats. In the present study, we further detected the effect of BMSCs co-expressing IL-18 and interferon (IFN)-β, both of which are immunostimulatory cytokines. BMSCs were genetically engineered to express IL-18 and IFN-β by transfection of recombinant lentivirus-mediated gene transfer. Results showed that BMSCs co-expressing the two cytokines displayed more significant inhibition effect on glioma cell growth in vitro when compared with BMSCs solely expressing IL-18 or IFN-β. Treatment of BMSCs co-expressing IL-18 and IFN-β significantly prolonged the survival and inhibited tumor growth in a rat intracranial glioma model. Furthermore, these genetically engineered BMSCs remarkably promoted cell apoptosis, antitumor cytokine production and CD4+ and CD8+ T-cell infiltration in intracranial glioma tissues than BMSCs solely expressing IL-18 or IFN-β. Results of the present study suggested that IL-18 and IFN-β had a synergistic effect on glioma inhibition. Moreover, results provided evidence that delivery of IL-18 and IFN-β by BMSCs may be an excellent and promising approach to develop an effective treatment protocol for glioma therapy.