The zinc finger transcription factor ZFX functions as an important regulator of self-renewal in multiple stem cell types, as well as a sex determinant of mammals. Moreover, ZFX expression is abnormally elevated in several cancers, and correlates with malignancy grade. To investigate its role in the pathogenesis of gliomas, we used lentivirus-mediated RNA interference (RNAi) to knockdown ZFX expression in human glioma cell lines. Our results demonstrate that ZFX plays a crucial role in glioma proliferation and survival, confirming recent reports. We also show for the first time that ZFX knockdown decreases the in vivo growth potential of U87 glioma xenografts in both subcutaneous and intracranial models in nude mice. We conclude that lentivirus-mediated RNAi targeting of ZFX may serve as a promising strategy for glioma therapy.