Hepatic stellate cells(HSC) in tumor microenviroment infiltrate the hepatocellular carcinoma(HCC) stroma, and they play a critical role in HCC progression. Epimorphin ( EPM, also called syntaxin2 ), a mesenchymal cell surface-associated molecule expressed in HSC, is a key regulator for liver progenitor cells differentiation,especially during the course of tubulogenesis. It has been reported that the dysfunction of EPM is related to ulcerative colitis, interstitial pneumonia and colon carcinoma. Therefore, the development of an EPM-knockdown HSCs will be highly beneficial in such studies. Stable EPM-knockdown transgenic cell lines were generated by transfecting human hepatic stellate cells with RNA interference(RNAi) plasmids. Reverse transcriptase polymerase chain reaction(PCR) analyses and Western blot indicated that the mRNA and protein levels of EPM in the transgenic cell lines were significantly lower than that in control. Conditioned media (CM) collected from the transgenic cell lines significantly decreased migration of HCC cells cultured in monolayers and transwells. HCCs and HSCs were co-cultured in 3D model, knocked-down EPM HSCs generated bigger spheroid culfures than control, the phenomenon also demonstrated that EPM could promote HCCs migration. The results show that RNAi can be used to stably knock down expression of EPM in human hepatic stellate cells and further improvements in related technologies will facilitate the studies of its roles in HCC tumorgenesis, proliferation and migration.