Objective To investigate the synergistic effect of gambogenic acid (GNA) and lenvatinib in inhibiting hepatocellular carcinoma (HCC) and promoting HCC apoptosis and related mechanism. Methods H22 hepatoma cells were used to establish a mouse xenograft tumor model, and after successful modeling, the mice were randomly divided into model group (normal saline), GNA group (20 mg/kg), lenvatinib group (10 mg/kg), and GNA (20 mg/kg)+lenvatinib (10 mg/kg) group, with 10 mice in each group. The mice in each group were given the corresponding drug by gavage once a day for 2 consecutive weeks. Tumor weight and body weight were compared between groups to observe the effect of GNA combined with lenvatinib on tumor growth; HE staining was used to observe the effect of GNA combined with lenvatinib on visceral organs in the mice with xenograft tumor; TUNEL staining was used to observe cell apoptosis in tumor tissue of the mice with xenograft tumor; Western blot was used to measure the protein expression levels of pregnane X receptor(PXR), P-glycoprotein(P-gp), breast cancer resistant protein(BCRP), cytochrome P450 3A4(CYP3A4),P53,B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax), and cleaved-caspase-3 in tumor tissue of the mice with xenograft tumor. Results Compared with the model group, GNA combined with lenvatinib significantly inhibited tumor growth (P<0.05) and had a better effect than GNA or lenvatinib used alone (P<0.05).After administration, no obvious lesions were observed in the major organs (heart, liver, spleen, lung, and kidney) of the mice. Compared with the model group, all administration groups showed significant cell apoptosis, and the GNA+lenvatinib group showed more significant cell apoptosis than the lenvatinib group. The GNA+lenvatinib group had significant reductions in the protein expression levels of PXR,P-gp,BCRP,CYP3A4, and Bcl-2 (P<0.05) and significant increases in the protein expression levels of P53,Bax, and cleaved-caspase-3 (P<0.05) in tumor tissue. Conclusion GNA combined with lenvatinib can exert a synergistic effect and enhance the sensitivity of HCC to lenvatinib, possibly by downregulating the expression of the nuclear receptor PXR and affecting the expression of the metabolic enzyme CYP3A4, the transporters P-gp and BCRP, and the apoptotic proteins P53,Bax,Bcl-2, and cleaved-caspase-3.