It has been hypothesized that cancer stem-like cells are responsible for tumor recurrence following chemotherapy. Evidence on the mechanisms through which drug-resistant stem-like cells recapitulate the tumor mass has not been definitively reported. Based on this information, we investigated the enrichment ability of a population of stem-like cells following treatment with cisplatin in human A549 cells and focused on the molecular mechanisms regulating the self-renewal of stem-like cells. A population of stem-like cells was enriched following cisplatin treatment and was defined phenotypically and functionally based on the expression of certain stem cell markers, sphere-forming ability, multipotent differentiation and induction of xenograft tumors in vivo. For various types of differentiated cells, Bmi1 has been reported to be important for cell proliferation and for the self-renewal of stem cells. The high expression of Bmi1 in cisplatin-enriched stem-like cells was shown using Q-PCR and western blotting; therefore, the role of Bmi1 was investigated in cisplatin-enriched stem-like cells by infecting cisplatin-enriched stem-like cells with Bmi1-targeted RNAi lentiviruses. Cell proliferation, tumor sphere formation and xenograft formation was reduced following knockdown of Bmi1. Based on our results, we propose that, after cisplatin treatment, Bmi1 is required for the self-renewal of stem-like cells that are important for the expansion of the stem-like cell pool in human A549 cells and that targeting Bmi1 slows down the formation of tumors in vivo.