Objective：In the present study，we aimed to investigate the small-molecule B lymphoma Mo-MLV insertion region 1 (Bmi1) inhibitor，PTC-209，for its effects on endogenous Bmi1 expression and cell behaviors in tongue squamous cell carcinoma (TSCC) cell line in vitro. Methods：The TSCC cell line Cal27 was treated with PTC-209 in vitro，and the expression change of Bmi1 was detected by Western blotting and real-time RT-PCR assays. The changes of cell proliferation，invasion and migration were analyzed by MTT，transwell and wound-healing assay，respectivety. The cancer stem cell (CSC) subpopulations in Cal27 were further assayed by tumorsphere formation，colony-forming and flow cytometry assays. Results：Bmi1 mRNA and protein levels were significantly decreased in Cal27 cells treated with PTC-209 in both dose- and time-dependent manners (P＜0.05). PTC-209 inhibited cell proliferation，migration and invasion，and had synergic anti-cancer effects when combined with cisplatin and 5-FU. Moreover，colony formation，sphere formation，and ALDH+ subpopulation were significantly reduced upon PTC-209 exposure. Conclusion：PTC-209 treatment induces Bmi1 reduction in TSCC cell line in vitro，and shows potent anti-tumor activity with reduced cell proliferation，migration，invasion and CSC subpopulation.