To elucidate biological functions of different protein Annexin1 discovered from hepatocellular carcimomar (HCC) cell lines by comparative proteomics approach. The different expression level of Annexin1 among HCC cell lines was further validated using other approachs including RT-PCR, Western blot and cell immunochemistry. The pcDNA3.1(+) AS Annexin1 expression plasmid was constructed, and then transfected into high metastatic cell lines MHCC97H to observe the alteration of biological functions of MHCC97H (motility, invasion, extracellular matrix metalloproteinase). All data from validation tests confirmed the overexpression of Annexin1 in high metastatic HCC cell lines with the comparison of non-metastatic one. Inhibition of the expression of Annexin1 in MHCC97H was successfully detected by RT-PCR after transfected with anti-sense RNA. According to analysis sequence followed as MHCC97H/pcDNA3.1(+) AS Annexin1, MHCC97H/ pcDNA3.1(+) and MHCC97H. Cell motility assay in vitro showed the average amount of invading cell per field were 11.13±3.31, 18.88±2.03, 21.86±3.39 respectively. The average amount of invading cell per field in cell invasion assay were 16.43±2.23, 16.4±1.57, 16.86±1.52 respectively.The Average clone formation ration of MHCC97H transfected was (14.33±0.46)%,(19.35±0.49)%, (20.25±0.35)%. The amount of apoptosis cell transfected by FCM analysis was 22.2%, 6.44%,6.97%. The number of transfected cell in each phase during cell cycles displayed G0-G1 phase 79.5%/ 76.34%/ 80.5%, S phase 13.26%/ 14.4%/9.69%, G2-M phase 7.25%/ 9.26%/ 9.81%. The concentration of MMP9 in serum free culture media by quantitative assay (ABC-ELISA) were 26.37 μg/L, 28.00 μg/L, 31.90 μg/L and MMP2 were 29.79 μg/L, 26.37 μg/L, 26.53 μg/L. The activities of MMPs in serum free culture media were detected by Gelatin zymography. No significant changes were found between target sample and control. Analysis of the results mentioned, some biological properties (including motility, clone formation potential) of MHCC97H transfected with pcDNA3.1(+) AS Annexin1 were all down-regulation, and yet the amount of apoptosis cell transfected were increased. No significant changes in cell invasion, cell cycle, MMPs were discovered. All the data suggested that Annexin1 may involved in HCC metastasis by alteration of cell apoptosis and of cell motility property.