B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) is directly involved in cell growth, proliferation and self-renewal of cancer stem cells (CSCs). The aim of the present study was to assess the role of Bmi1 in the maintenance of stemness properties and tumorigenicity of human bladder CSC-like side population (SP) cells. SP cells were sorted by flow cytometry using Hoechst 33342 staining. Bmi1 mRNA and protein expression in SP and non-SP (NSP) cells was analyzed by quantitative PCR, immunofluorescence and western blotting. The stemness properties of SP cells included cell proliferation, migration, self-renewal, chemotherapy resistance and cell cycle progression were assessed. Tumor formation was also assessed in human bladder cancer xenografts after Bmi1 silencing. The mRNA expression of Bmi1 was upregulated in SP cells when compared with that in the NSP cells. Knockdown of Bmi1 in SP cells resulted in inhibition of cell proliferation, migration and tumor sphere formation, enhanced sensitivity to cisplatin, and cell cycle arrest in the G0/G1 phase. Bmi1 knockdown inhibited cell cycle progression through derepression of the p16INK4a/p14ARF locus. Bmi1-siRNA SP cells failed to produce tumors in recipient mice, while typical urothelial carcinoma formed from subcutaneously injected scramble-siRNA SP cells. Bmi1 is crucial for the maintenance of stemness properties and tumorigenicity of human bladder CSC-like cells. Bmi1 may be a potential therapeutic target for the eradication of CSCs in bladder cancer.