Abstract: Objective: To investigate the anti-tumor activity and potential mechanism of the combination therapy of Anoectochilus roxburghii polysaccharides (ARP-0II) and vitamin C (V_C) on human liver cancer HepG2 cells. Methods: The crude polysaccharide of Anoectochilus roxburghii was isolated and purified by ion exchange column and dextran gel column. The purified polysaccharide ARP-0II was obtained, and its structure was characterized by methylation Gas Chromatography-Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR). Using HepG2 cells as a model, MTT assay was used to detect cell proliferation inhibition rate and flow cytometry was used to analyze cell cycle and apoptosis; Western blot (WB) was used to detect the expression levels of apoptosis related proteins (Bax, Bcl-2, Caspase 7) and CompuSyn software was used to evaluate the synergistic effect of combination therapy. Result: ARP-0II was mainly composed of glucose residues, with a structure of →4)-α-D-Glcp-(1→ as the main chain and containing a small number of branches. ARP-0II and V_C both had concentration dependent inhibitory effects on HepG2 cells, with IC₅₀ values of 1281 μg/mL and 52.28 μg/mL, respectively. The combination therapy showed a synergistic effect (CI<1), with a maximum inhibition rate of 70.75%. Cell cycle analysis showed that drugs arrested cells in the S phase; The apoptosis experiment showed that the combination therapy significantly increased apoptosis rates in the early and late stage of cell cycle; Protein expression analysis indicated that its apoptosis mechanism might be related to the mitochondrial pathway. WB results showed that ARP-0II and Vc could regulate the expression of apoptosis-related proteins (Caspase 7, Bax, Bcl-2), and the combination could significantly（P<0.05）up-regulate the levels of Caspase 7 and Bax and inhibit the expression of Bcl-2, suggesting that the apoptosis mechanism may be related to the mitochondrial pathway. Conclusion: The combination of Anoectochilus roxburghii polysaccharides and Vc had a synergistic anti-tumor effect on HepG2 cells, and its mechanism might be related to inducing cell cycle arrest, promoting cell apoptosis, and regulating the expression of apoptosis related proteins. This study provides experimental evidence and theoretical basis for the application of natural product combination therapy in the prevention and treatment of liver cancer.