OBJECTIVE To detect the expression of CD133 in human tongue squamous cell carcinoma Tea8113 cell line and observe proliferation ability of CD133 groups in vitro. METHODS Limiting dilution assay was employed to observe the proliferating character of Tca8113 single cell in vitro. The ability of growing as cancer spheroids was observed with ultra-low attachment plates. The flow cytometry was used to detect the expression of putative tumor-initiating cell marker CD133 in human tongue squamous cell carcinoma Tca8113 cell line. The selective technique of immunomagnetic beads was applied to purify CD133 tumor cells, CD133 tumor cells were cultured and their ability of proliferation were observed in vitro. RESULTS After 12 days, the result of single cell culture in vitro revealed that about 5.23% of cultured Tca8113 cells possessed the capacity of continue proliferation. The cells line fromed floating clusters with one week of passaging cells into non-adherent plates. Approximately 0.95% of cells in Tca8113 cell line expressed CD133. Compared with CD133- cells and control Tca8113 cells, CD133+ cells demonstrated increased proliferation capacity. The proportion of CD133 cells decreased in culture as days passed. The percentage of CD133+ cells decreased from 92.45% to 1.62% in twelve days' culture. CONCLUSIONS Tumor stem cells have the character of heterogenity and lower proportion of CD133 but higher ability of proliferation, and the diferentiation in human tongue squamous cell carcinoma Tca8113 cell line in vitro, CD133 may be one of makers for tumor-initiating cell of human tongue squamous cell carcinoma Tca8113 cell line.