Control of the cancer stem/initiating cell population is considered key to realizing the long-term survival of glioblastoma patients. Recently, we demonstrated that FOXO3 activation is sufficient to induce differentiation of glioma-initiating cells having stem-like properties and inhibit their tumor-initiating potential. Here we identified metformin, an antidiabetic agent, as a therapeutic activator of FOXO3. Metformin activated FOXO3 and promoted differentiation of such stem-like glioma-initiating cells into nontumorigenic cells. Furthermore, metformin promoted FOXO3 activation and differentiation via AMP-activated protein kinase (AMPK) activation, which was sensitive to extracellular glucose availability. Importantly, transient, systemic administration of metformin depleted the self-renewing and tumor-initiating cell population within established tumors, inhibited tumor formation by stem-like glioma-initiating cells in the brain, and provided a substantial survival benefit. Our findings demonstrate that targeting glioma-initiating cells via the AMPK-FOXO3 axis is a viable therapeutic strategy against glioblastoma, with metformin being the most clinically relevant drug ever reported for targeting of glioma-initiating cells. Our results also establish a novel, direct link between glucose metabolism and cancer stem/initiating cells.