Abstract: Objective To investigate the influence of Adriamycin (ADM) on human hepatocellular carcinoma(HCC) cells with different metastatic abilities and related gene expressions of HCC stem cell. Methods MTT was used to detect the inhibition effect of ADM on human HCC cell lines MHCC97-L and HCCLM3, to compute the LD50 concentration. Western blot was used to detect the expression levels of Nanog, Oct-4, Sox2, ARID1 and Wnt5b in human HCC cell lines MHCC97-L and HCCLM3, and to analyze the difference of their expression levels between the two different cell lines. Results With the increased ADM level, the inhibition effect was improved gradually, and the LD50 to MHCC97-L and HCCLM3 cell lines were 0.4212 and 0.5249μmol/L, respectively. As the action time of ADM (LD 50) prolonged, the level of Wnt5b was raised at first and then declined, while the level of Nanog was declined at first and then raised in both MHCC97-L and HCCLM3 cell lines. The level of Sox2 was improved in HCCLM3 cells in a time-dependent manner. The levels of Wnt5b and Nanog were lower in MHCC97-L cells than those in HCCLM3 cells within 4h (P<0.05). Conclusion ADM could promote the apoptosis rate of MHCC97-L and HCCLM3 cell lines, and the influence was much better in MHCC97-L cells than that in HCCLM3 cells; Wnt5b is negatively correlated with the tumors malignancy degree; Nanog and Sox2 are closely related with the metastasis of the tumor, which may related with the drug resistance of HCC.