The objective of this study is to investigate the chemoresistance of CD133(+) cancer stem cells in Hep-2 cells of laryngeal cancer and detect the expression mRNA and protein levels of BMI-1 in CD133(+) cells and CD133(-) cells. The response of Hep-2 cells to different chemotherapeutic agents was investigated, and the expression of CD133 was studied. Fluorescence-activated cell sorting analysis was used to identify CD133, and the CD133(+) subset of cells was separated and analyzed chemotherapy resistance. Colony formation assays were studied and cells were injected subcutaneously into axillary fossa of node mice to measure the tumor-forming ability. RT-PCR and Western blot analyses were used to detect the expression levels of BMI-1 in the different subpopulation cells. It was concluded that chemotherapy enriched the CD133(+) subpopulation 2-fourfold, relative to the untreated cells. 1.55 ± 0.28% of Hep-2 cells were observed to be CD133(+) cells. Flow cytometric analysis revealed that after the treatment with these chemotherapeutic agents, the expression of CD133 was up to 5.16 ± 0.86%, 4.94 ± 0.58%, 3.66 ± 0.59%. After 5-FU treatment, the expression of CD133 was 6.7 ± 1.6% relative to the untreated mice 2.6 ± 0.96% by nude mice tumor xenograft model. CD133(+) cancer stem cells were more resistant to chemotherapy; the proliferation capability and tumor-forming ability were no difference after chemotherapy. Semi-quantitative RT-PCR and Western blot analyses provided strong evidence that BMI-1 expression in CD133(+) cells is different from CD133(-) cells remarkably. Taken together, it was confirmed that CD133(+) cancer stem cells were chemoresistant and BMI-1 was highly expressed in these CD133(+) cells.