Objective: To investigate the influence of downregulating CDl33 gene expression on biological behaviors of human liver cancer cells. Methods: Liver cancer SMMC7721 cells were transfected with the synthesized CDl33 siRNA and then transfection efficiency was determined. Using SMMC7721 cells without transfection or transfected with scrambled siRNA sequence as blank control and negative control respectively, the effect of CD133 gene silencing, and changes in the main biological behaviors in SMMC7721 cells after CDl33 siRNA transfection were observed. Results: The transfection efficiency reached (80.8±9.1) % at 24 h after transfection. Compared with blank control group, in SMMC7721 cells after CDl33 siRNA transfection, the expression level of CDl33 mRNA and protein was reduced to 10% and 35% of the level in blank control group respectively, the proliferative activity was significantly decreased, apoptosis rate was significantly increased with a marked S-phase arrest, and colony-forming ability was significantly decreased (all P<0.05). All indexes did not show any significant difference between negative control group and blank control group (all P>0.05). Conclusion: CDl33 gene may play an oncogene role in liver cancer cells and down-regulating its expression can suppress the malignant activity of liver cancer.