We employed lentivirus-based doublecortin (DCX), as a glioma suppressor gene therapy in an intracranial glioma tumor xenograft model in nude rats. Single DCX-expressing lentivirus was directly administered into the tumor on day 8 after U87 tumor cell implantation. DCX treatment significantly reduced U87 glioma tumor volume (∼60%) on day 14 after DCX lentivirus injection and significantly improved median survival of tumor-bearing nude rats. DCX synthesis induced neuronal markers MAP2, TUJ1, and PSA-NCAM and the glial marker glial fibrillary acidic protein (GFAP) in the implanted U87 glioma tumors. DCX synthesis induced GFAP that colocalized with tubulin in the mitotic stage, inhibited cleavage furrow during cytokinesis, and blocked mitosis in glioma cells. DCX lentivirus infection did not induce apoptosis but significantly inhibited expression of the proliferation marker Ki-67 and the blood vessel marker von-Willebrand factor (vWF). U87 and other glioma cells except for brain tumor stem cells (BTSCs) do not express neuronal markers or both neuronal and glial markers. DCX-synthesizing glioma cells express a phenotype of antiangiogenic BTSC-like cells with terminal differentiation that causes remission of glioma cells by blocking mitosis via a novel DCX/GFAP pathway. Direct local delivery of lentivirus-based DCX gene therapy is a potential differentiation-based therapeutic approach for the treatment of glioma. © 2009 Wiley-Liss, Inc.