Objective:To investigate both mRNA and protein levels of B lymphoma Mo-MLV insertion region 1 (Bmi1) in a panel of tongue squamous cell carcinoma (TSCC) cell lines as compared with normal tongue mucosa, and then to study biological roles of Bmi1 in tongue tumorigenesis by loss-of-function assays using small interference RNA. Methods: RNA and protein expressions of Bmi1 in TSCC cell lines and normal tongue mucosa were detected by qRT-PCR and Western blot. Cellular immunofluorescence was performed to further characterize the subcellular distribution of Bmi1 in tongue cancer cell lines. Cell migration, invasion, proliferation and colony formation were assessed by wound-healing, Transwell, MTT and colony-forming experiments. To further reinforce the notion that Bmi1 is critical for tongue cancer growth in vivo, genetic approach was further utilized to inhibit Bmi1 in a tongue cancer xenograft model. Results: Bmi1 mRNA and protein levels in TSCC cell lines were significantly higher than that in normal tongue mucosa as assessed by real-time RT-PCR and Western blot assays (P < 0.05). Short-hairpin RNA-mediated Bmi1 knockdown inhibited cell proliferation, migration and invasion, reduced colony formation, presumably by modulation of p16, p14 and E-cadherin. Short-hairpin RNA-mediated Bmi1 knockdown significantly impaired tumor growth in a tongue cancer xenograft model. Conclusion:Bmi1 may serve as a key driver with multiple biological functions during tongue cancer progression and a novel therapeutic target against tongue cancers.