Glioblastoma (GBM) are characterized by an infiltrative nature and high resistance to radio- and chemotherapy. Despite advances in novel therapeutic approaches, the median survival of 12-14 months has not changed significantly. Therefore, there is an imperative need for the identification of alternative therapeutic approaches. The anti-diabetic biguanide drug metformin has been reported to exert anti-tumor effects. Phenformin, a metformin analog, is a more potent inhibitor of tumor growth, especially for Liver Kinase B1-deficient tumors. Our study demonstrates, for the first time, the effect of phenformin on the stemness and apoptosis of glioma cells and glioma stem cells (GSCs). We found that phenformin inhibited the self-renewal of GSCs, decreased the expression of the stemness markers, CD44 and Sox2 and the mesenchymal transformation of these cells. Phenformin at higher concentrations also induced apoptosis in both GSCs and differentiated glioma cells. The expression of AMPK and the phosphorylation of this kinase in response to phenformin were relatively low in GSCS, suggesting that this drug impacted the self-renewal and apoptosis of these cells mostly in an AMPK-independent manner. Phenformin inhibited the STAT3 pathway and its effects on GSCs were further enhanced by STAT3 inhibitors. In addition, phenformin decreased the expression of the long non-coding RNA, H19 and increased the expression of miR-124 and Let-7 and silencing of these miRNAs abrogated phenformin effects. Combined treatment of phenformin with radiation and temozolomide exerted a synergistic anti-tumor effects on GSCs which were more significant than those exerted by combined treatments with metformin. Using GSC-derived xenografts we demonstrated that phenformin inhibited tumor growth and angiogenesis, increased cell apoptosis and prolonged the overall survival of mice bearing GSC-derived xenografts. Our results demonstrate that phenformin can target GSCs via the STAT3/H19/Let7 axis and can be efficiently combined with current therapies for the treatment of GBM and the eradication of GSCs.