Gliomas account for approximately 80% of primary malignant tumors in the central nervous system. Despite aggressive therapy, the prognosis of patients remains extremely poor. Glioma stem cells (GSCs), considered a potential target of therapy for their crucial role in therapeutic resistance and tumor recurrence, are believed to be key factors in the disappointing outcome. Here, we took advantage of GSCs as the cell model to perform high-throughput drug screening, and the old antibiotic clofoctol was identified as the most effective compound, showing reduction of colony formation and induction of apoptosis of GSCs. Moreover, growth of tumors was obviously inhibited in vivo after clofoctol treatment especially in primary patient-derived xenografts and transgenic xenografts. The anticancer mechanisms demonstrated by analysis of related downstream genes and discovery of the targeted binding protein revealed that clofoctol exhibited the inhibition of GSCs by upregulation of Kruppel-like factor 13 (KLF13), a tumor suppressor gene, through clofoctol's targeted binding protein, Upstream of N-ras (UNR). Collectively, these data demonstrate that induction of KLF13 expression suppressed growth of gliomas and provide a potential therapy for gliomas targeting GSCs. Importantly, our results also identify the RNA-binding protein UNR as a drug target.