Objective To establish a method for isolating and purifying tumor-associated hepatic stellate cells (tHSCs) from human hepatocellular carcinoma (HCC) tissues and to study their impact on the biological behaviors of HCC cells in vitro. Methods The tHSCs were isolated from the fresh human HCC tissue by collagenase/pronase digestion, followed by density gradient centrifugation using Nycodenze. The viability of the isolated cells was determined by trypan blue staining assay. The tHSCs were identified by immunocytochemical staining of α-SMA, a specific marker for activated hepatic stellate cells. The morphologic changes of the tHSCs were observed under microscope. The conditioned medium (CM) of tHSCs cultured in serum-free DMEM was collected, and then used for co-culture with HCC cell lines QGY-7701, BEL-7402, and SMMC-7721. CM of human hepatic stellate cell line LX-2 was used as control. Cell viability, proliferation, migration potential and EMT-related protein expression changes were assessed by using the MTT assay, wound migration assay and Western blotting analysis, respectively. Results The viability of isolated tHSCs was over 95%, with a purity about 100%, and the isolated tHSCs could be passaged and cryopreserved. tHSCs significantly induced typical EMT-like morphological changes and altered expression of molecular markers in the three HCC cell lines, with the epithelial marker E-cadherin significantly decreased (P<0.05) and the stromal marker Vimentin significantly increased (P<0.05). Meanwhile, tHSCs efficiently enhanced the proliferation and migration of HCC cells in vitro. Conclusion We have established a method for isolating and purifying tHSCs from human HCC tissues, which can help future study on tHSCs. tHSCs can enhance the proliferation and migration of HCC cells through inducing EMT-like phenotype.