Objective To study the inhibitory effects of gentiopicroside on tumor growth and anti-angiogenesis in mice with H₂₂ hepatocellular carcinoma. Methods A H₂₂ hepatocellular carcinoma mouse model was established by subcutaneous injection of a H₂₂ cell suspension into the right axilla. Mice with H₂₂ hepatocellular carcinoma were randomly divided into a model group, low and high dose gentiopicroside groups ( 50 and 100 mg / kg, respectively ), and a cyclophosphamide group (20 mg / kg) with 10 mice in each group. Another 10 healthy mice were used as the normal group. After 14 days of administration, body weight, tumor weight, tumor inhibition rate, thymus index, spleen index, serum IFN-γ and IL-2 contents, and expression levels of bFGF, TGF-β, VEGF, p-PI3K, and p-Akt were measured in tumor tissues. Results Compared with the H₂₂ hepatocellular carcinoma model group, the body weights of mice in low and high dose groups of gentiopicroside did not change significantly (P>0. 05), while the tumor weight was reduced significantly (P<0. 01),with the tumor inhibition rates of 30. 43% and 42. 93%, respectively. Compared with the H₂₂ hepatocellular carcinoma model group, the thymus index, spleen index, and serum IFN-γ and IL-2 contents of mice in low and high dose gentiopicroside groups were increased significantly (P<0. 05 or P<0. 01), while expression of bFGF, TGF-β, VEGF, p-PI3K, and p-Akt in tumor tissues was decreased significantly (P<0. 05 or P<0. 01). Conclusions Gentiopicroside has an inhibitory effect on tumor growth and anti-angiogenesis effect in H₂₂ hepatocellular carcinoma mice, which is related to improving immunity, increasing serum IFN-γ and IL-2 levels, and inhibition of activation of the PI3K/ Akt signaling pathway.