Betulinic acid (BA) is a pentacyclic triterpenoid compound that widely exists in Chinese herbal medicine, and it has remarkable biological activity. However, the involved molecular targets and mechanisms of BA are still ambiguous. Here, we aim to validate the preventive effects and molecular mechanisms of BA against hepatocellular carcinoma via related experiments. We extracted the 2D and 3D structure of BA from the PubChem database. MTT assay and colony formation assay were used to determine the anti-proliferation and cytotoxicity of BA using in vitro cell models. Hoechst 33258 staining was used to investigate the extent of apoptosis after BA treatment. Western blot and immunofluorescence experiments were used to evaluate apoptosis-related and autophagy-related proteins and molecular mechanisms. We demonstrated that BA significantly inhibited cell proliferation in HepG2 and SMMC-7721 hepatocellular carcinoma cells, but with little cytotoxicity effects on l-02 normal liver cells. We further determined that the hepatocellular carcinoma prevention effects of BA were closely correlated with apoptosis and autophagy. Furthermore, our data indicated that BA-induced autophagy has a protective effect against cancer cell proliferation and promotes cell apoptosis. Additionally, apoptosis and autophagy were induced by BA through suppression of the PI3K/AKT/mTOR signaling pathway. Collectively, our study provides experimental evidence that BA inhibits cell proliferation and induces cell apoptosis and autophagy via suppressing the PI3K/AKT/mTOR pathway. Additionally, BA is a safe and effective herbal medicine compound that can be used for the prevention of hepatocellular carcinoma growth, and may be a potential therapeutic strategy against hepatocellular carcinoma. AJTR Copyright © 2019.