CD133 is a useful putative marker of cancer stem cells (CSCs) in human laryngeal tumors. Numerous studies have demonstrated that CD133+ CSCs possess higher clonogenicity, invasiveness and tumorigenesis compared with CD133- cells. Recently, interest in the Warburg effect in the microenvironment of CSCs has escalated. The Warburg effect dictates that cancer cells rely on glycolysis rather than oxidative phosphorylation under aerobic conditions. In numerous cancer cells, glucose is used mainly for the glycolytic pathway. Stem cells express high levels of glycolytic enzymes and rely mostly on glycolysis to meet their energy demands. Glucose is transported through cell membranes by glucose transporters (Glut). Studies of Glut-1 expression in CSCs are limited. In the present study, we investigated the proliferation of CD133+ Hep-2 cells and whether Glut-1 is expressed in laryngeal carcinoma CD133+ Hep-2 cells. Real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that the size of the CD133 product was 213 bp. Dissociation curve analysis demonstrated only the expected peaks at 82.1˚C for CD133. The mean ΔCt of CD133 expression was 10.98. Prior to isolation, the CD133+ fraction was 1.2% by fluorescence-activated cell sorting (FACS) analysis. Following isolation, the CD133+ fraction was increased to 76.1%. Successive tests also demonstrated that cells grew well following isolation. The proliferation of CD133+ and CD133- cells was not different during the first 3 days (P>0.05). From day 4, the proliferation capacity of CD133+ cells in vitro was higher than that of CD133- cells (P<0.05). The mean ΔCt of Glut-1 mRNA expression was 1.78 for CD133+ cells and 1.00 for CD133- cells (P<0.05). The mean Glut-1 protein values in CD133+ and CD133- Hep-2 cells relative to β-tubulin were 0.48 ± 0.02 and 0.21 ± 0.03 (µg/µl), respectively (P<0.05). In conclusion, CD133+ cells demonstrated higher proliferation. Glut-1 mRNA and protein levels were higher in CD133+ than in CD133- cells. Our results suggest that Glut-1 is important in the energy supply of laryngeal CD133+ Hep-2 cells and Glut-1 may represent a potential therapeutic target for the inhibition of the proliferation of laryngeal CSCs.