Abstract: Objective to observe effects of AFP gene silencing by siRNA on proliferation of hepatocellular carcinoma cell line EGHC-9901. Methods siRNA expressing plasmid aimed at AFP gene was firstly established and subsequently transfected into hepatocellular carcinoma cell line EGHC-9901; after G418 positive clone selection for 4-5 weeks, AFP expression both of mRNA and protein were detected by semi-quantitative RT-PCR and western blot; cells then were divided into three groups: experimental group, AFP-siRNA transfected; vector control group, empty vector transfected; blank group, untreated. MTT and flat plate clone formation assay were applied to evaluated cell proliferation, and flow cytometry was employed to observe cell cycle. Results siRNA expressing plasmid aimed at AFP gene was successfully transfected into hepatocellular carcinoma cell line; AFP expression was almost completely inhibited. The experimental group proliferated at a significantly lower speed than the positive control group; The experimental group exhibited less clones with diameters more than 75μm than the positive control group, p<0.05; flow cytometry manifested approximately 20% more cells of the experimental group within G1 phase than those of the positive group, indicating that inhibition of AFP expression may cause G1 phase arrest. Conclusion siRNA expressing plasmid aimed at AFP gene was successfully stably transfected into hepatocellular carcinoma cell line EGHC-9901; AFP gene silenced by siRNA induces growth and colony formation inhibition of hepatocellular carcinoma cell line EGHC-9901.