Objective To observe the effects of betulinic acid (BA) on proliferation of human hepatoma stem cell;To discuss its anti-cancer mechanism from the aspects of cell cycle and cell apoptosis. Methods HepG2 stem cells were cultivated in vitro and testified the self-renewal capacity. The effects of BA in concentration of 40, 20, 10, 5, 2.5, 1.25μmol/L on the cell vitality of cultured human liver cancer stem cells for 24 and 48 hours were measured with CCK-8 method. The human hepatoma stem cell line HepG2 was administrated by BA at concentrations of 40, 20, 10, 5μmol/L for 48 hours, and cell cycle and apoptosis rate were measured by flow cytometry. Results BA could inhibit HepG2 stem cell proliferation obviously with dose-effect relationship. BA influenced cell cycle, and induced tumor stem cell apoptosis. 40μmol/L BA blocked cell cycle in S phase, and cell apoptosis rate reached 10.86%. Conclusion BA has obvious inhibitory effects on proliferation of HepG2 liver cancer stem cell, which probably plays a part in anti-cancer by influencing cell cycle and inducing cell apoptosis.