Immunotoxins have shown great promise as an alternative treatment for brain malignancies such as gliomas, but their failure to penetrate into the tumor mass remains a major problem. Mesenchymal stem cells exhibit tropism to tumor tissue and may serve as a cellular vehicle for the delivery and local production of antitumor agents. In this study, we used human bone marrow-derived mesenchymal stem cells (hMSCs) as a vehicle for the targeted delivery of EphrinA1-PE38, a very specific immunotoxin against the EphA2 receptor that is overexpressed in gliomas. hMSCs were transduced with adenovirus to express secretable EphrinA1-PE38. Our in vitro assays confirmed the expression, release and selective killing effect of the immunotoxin produced by hMSCs. Furthermore, the intratumoral injection of engineered hMSCs was effective at inhibiting tumor growth in a malignant glioma tumor model. These results indicate that gene therapy utilizing EphrinA1-PE38-secreting hMSCs may provide a novel approach for the local treatment of malignant gliomas.