Recent studies suggest that a small subpopulation of malignant cells with stem-like properties is resistant to chemotherapy and may be responsible for the existence of residual cancer after treatment. We have isolated highly tumorigenic cancer cells with 100-fold increase in tumor initiating capacity from the tumor xenografts of human glioblastoma U87 cells in mice. These cells exhibit stem-like properties and show unique energy metabolic characteristics including low mitochondrial respiration, increased glycolysis for ATP generation, and preference for hypoxia to maintain their stemness and tumor forming capacity. Mechanistically, mitochondrial depression in the highly tumorigenic cells occurs mainly at complex II of the electron transport chain with a down-regulation of the succinate dehydrogenase subunit B, leading to deregulation of hypoxia-inducible factors. Under hypoxia, the stem-like cancer cells are resistant to conventional anticancer agents but are sensitive to glycolytic inhibition. Furthermore, combination of glycolytic inhibition with standard therapeutic agents is effective in killing the tumor-initiating cells in vitro and inhibits tumor formation in vivo. Our study suggests that stem-like cancer cells prefer a low oxygen microenvironment and actively utilize the glycolytic pathway for ATP generation. Inhibition of glycolysis may be an effective strategy to eradicate residual cancer stem cells that are otherwise resistant to chemotherapeutic agents in their hypoxic niches.