In this study, we investigate the effect of short hairpin RNA-mediated gene silencing of Bmi-1 expression on chemosensitivity of CD44+ nasopharyngeal carcinoma cancer stem-like cells. The sequence-specific short hairpin RNA lentivirus targeting at human Bmi-1 was synthesized and used to infect CD44+ nasopharyngeal cells that were sorted by flow cytometry. We also employed flow cytometry to detect transfection efficiency. Real-time polymerase chain reaction was used to detect Bmi-1 and its downstream repressor genes p16INK4a and p14ARF messenger RNA, while each protein expression level of Bmi-1, p16INK4a, p14ARF, and p53 was confirmed by Western blotting protocol. Tumor spheroid assay was used to evaluate the self-renewal capacity. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and colony formation assay were applied to detect proliferation capacity and colony-forming capacity under different concentrations of chemotherapeutic drugs 5-fluorouracil or cisplatin. Transwell cell migration and invasion assay were employed to observe migration and invasion capacity after cells were exposed to cisplatin for 24 hours. The constructed short hairpin RNA lentivirus targeting Bmi-1 gene successfully infected into the CD44+ nasopharyngeal carcinoma cells and effectively inhibited the Bmi-1 messenger RNA and protein expression level, while the expression level of Bim-1 target genes, p16INK4a, p14ARF, and p53 was significantly increased (P < .05). Notably, the proliferation, colony formation, migration, and invasion capabilities of the sequence-specific short hairpin RNA lentivirus-infected CD44+ nasopharyngeal carcinoma cells reduced significantly under chemotherapeutic treatments (P < .05). Our results indicated that Bmi-1 may play an important role in the chemosensitivity of CD44+ nasopharyngeal carcinoma cancer stem-like cells. Bmi-1 may be a potential new target for the treatment of nasopharyngeal carcinoma displaying chemotherapy resistance.