Despite aggressive multimodal therapy and advances in imaging, surgical and radiation techniques, high-grade gliomas remain incurable, with patient survival often measured in months. Treatment failure is largely attributable to the invasive nature of glioma cells, ineffective delivery of chemotherapeutic agents across the blood-brain barrier, and dose-limiting systemic toxicities. Neural stem cells (NSCs) have inherent tumor-tropic properties that can be exploited for targeted delivery of anti-cancer agents. However, current intracranial and intravenous injection approaches for administering NSCs are not optimal, especially for repeat administrations, because the methods are invasive and may lead to complications. We hypothesized that intranasal administration of NSCs would circumvent these challenges. In this study, we evaluated the biodistribution of NSCs administered intranasally to severely immunodeficient esterase-deficient Esle mice bearing orthotopic xenografts of U251.eGFP.ffluc human gliomas. Histological imaging and 3D reconstruction revealed that NSCs specifically localized to tumor sites, but not to non-tumor areas of the brain. Importantly, mice treated with intranasally administered NSCs that were genetically modified to express carboxylesterase, a prodrug-activating enzyme, in combination with CPT-11 showed increased survival and reduced tumor growth. These results support further development of intranasal delivery of NSCs as a new approach to treating glioma and possibly other invasive brain tumors.