Previously, we have shown that the genetically modified human neural stem cells (NSCs) show remarkable migratory and tumor-tropic capability to track down brain tumor cells and deliver therapeutic agents with significant therapeutic benefit. Human NSCs that were retrovirally transduced with cytosine deaminase (CD) gene showed remarkable 'bystander killer effect' on the glioma cells after application of the prodrug, 5-fluorocytosine (5-FC). Interferon-beta (IFN-beta) is known for its antiproliferative effects in a variety of cancers. In our pilot clinical trial in glioma, the IFN-beta gene has shown potent antitumor activity in patients with malignant glioma. In the present study, we sought to examine whether human NSCs genetically modified to express both CD and IFN-beta genes intensified antitumor effect on experimental glioma. In vitro studies showed that CD/IFN-beta-expressing NSCs exerted a remarkable bystander effect on human glioma cells after the application of 5-FC, as compared with parental NSCs and CD-expressing NSCs. In animal models with human glioma orthotopic xenograft, intravenously infused CD/IFN-beta-expressing NSCs produced striking antitumor effect after administration of the prodrug 5-FC. Furthermore, the same gene therapy regimen prolonged survival periods significantly in the experimental animals. The results of the present study indicate that the multimodal NSC-based treatment strategy might have therapeutic potential against gliomas.