Abstract: Objective To investigate the effects and the mechanism of co-stimulatory molecule B7-H3 on tumor metastasis of hepatocellular carcinoma （HCC）. Methods The B7-H3 expressions of surgically resected specimens in 37 pairs of HCC patients including 37 metastatic HCC cases and 37 cases without metastasis were confirmed by immunohistochemistry. The shRNA silencing plasmid was designed and transfected into HepG2 cells to down-graduate B7-H3 gene expression. The mRNA and protein expressions of EMT-related molecules including E-cadherin， vimentin and N-cadherin were detected by RT-PCR and Western blotting， separately. Results The expression of B7-H3 in tumor margin and metastases was higher than that of internal primary lesions. The B7-H3 expressions of primary lesions in the group with metastasis were much higher than which in the group without metastasis （P=0.01）. The mRNA and protein expression of E-cadherin in B7-H3 shRNA group were 1.27±0.23 and 1.03±0.27， which were higher than those of scramble shRNA group （0.71±0.16，0.80±0.05） and control group （0.63±0.11，0.71±0.09）. The difference was statistically significant （P＜0.05）. The mRNA and protein levels of vimentin in B7-H3 shRNA group were 0.31±0.14 and 0.36±0.06， which were lower than scramble shRNA group（0.79±0.09，0.81±0.15） and control group（0.82±0.04，0.98±0.15）. The difference was statistically significant （P＜0.05）. The mRNA and protein levels of N-cadherin in B7-H3 shRNA group were 0.68±0.09 and 0.56±0.16， which were lower than scramble shRNA group（1.28±0.26，0.86±0.09） and control group（1.42±0.17，1.02±0.11）.The difference was statistically significant （P＜0.05）. Conclusion Costimulatory molecule B7-H3 could promote tumor metastasis of HCC， which might be achieved by regulating the EMT mechanism.