Abstract： Objective To investigate the in vitro anti-cancer effects of ouabin combined Na~+/K~+-ATPase α1 siRNA upon HepG2 cells and its molecular mechanism. Methods The Na~+/K~+ -ATPase α1 subunit expressions of human hepatoma tissue, normal liver tissue and human hepatoma cell lines (HepG2, SMC7721 and Bel7402) were determined. The cells received different treatments (0. 03 μmol/L siRNA, 0. 1 μmol/L ouabain and combination). The proliferation of HepG2 was observed by CCK-8 assay. The activity of Na~+/K~+-ATPase was measured. The HepG2 cell cycle distribution was detected by flow cytometry. The mRNA and protein levels of Na~+/K~+-ATPase α1 subunit, MAPK1, CyclinA, CDK2, PCNA and P21WAF1 were detected by quantitative RT-PCR and Western blot. Results The gray value Na~+/K~+ -ATPase α1 subunit in hepatoma tissue was 174. 74 ± 16. 77 and 65.31 ± 7. 88 respectively in normal liver tissue. The Na~+/K~+ -ATPase α1 subunit expression of hepatoma tissue was significantly higherthan normal liver tissue(P < 0. 05). The Na~+/K~+ -ATPase α1 subunit expression level of HepG2 was higher than that in SMMC-7721 and Bel-7402 cells. The CCK-8 experiments demonstrated that siRNA, ouabain and combination could inhibit the HepG2 proliferation, and the combination group was different from the ouabain or siRNA group (P < 0. 05). The 24, 48 and 72 h inhibitory rates of 0. 03 μmol/L siRNA, 0. 1 μmol/L ouabain and combination group were (17.4%, 20. 3%, 24. 3%), (37. 5%, 44. 3%, 51.2%) and (52. 3% ,70. 2% ,88. 2%) respectively. The activity of Na~+/K~+ -ATPase decreased in siRNA, ouabain and combination group. The S phase proportion of ouabain and combination group increased from 24. 2% to 66. 5% and 75. 2% respectively. The 0. 1 μmol/L ouabain and combination group could down-regulate the expression of Na~+/K~+-ATPsae α1, MAPK1, CyclinA, CDK2, PCNA and up-regulate the expression of P21WAF1 in HepG2 cell. Conclusion The Na~+/K~+ -ATPase α1 siRNA combined ouabain inhibits the proliferation of HepG2 cells by decreasing the expression of MAPK1 and induces the cell cycle S arrest by decreasing the production of CyclinA/CDK2/PCNA complex and increasing the expression of P21~(WAF1).