Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.