To study the antitumor effect of glycyrrhiza polysaccharide (GPS) on human hepatocellular carcinoma cells and its mechanism, GPS was extracted and identified with phenol-sulfuric acid assay, Limulus amebocytes lysate assay, gel permeation chromatography, and infrared spectroscopy analysis. To study its antitumor function, 4-5-week-old imprinting control region mice were subcutaneously implanted with H22 cells and intragastrically subjected to 1 ml GPS (25, 50, and 75 mg/kg/day), 150 mg/kg cyclophosphamide in a dose of 150 mg/kg, or equal volume of phosphate buffered saline as control. Tumor weights were detected 10 days later. Apoptosis of intraperitoneally cultured and GPS-treated H22 cells was identified by flow cytometry and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide. In vitro, the function of GPS on cell proliferation was applied on BEL7402 cells and confirmed by 4,6-diamidino-z-phenylindole staining. Assessment of the effect of GPS on P53 gene was analyzed by real-time PCR and Western blot, and the effects of GPS on phosphatidylinositol-3 kinase (PI3K), AKT, p-PI3K, and p-AKT were analyzed by Western blot. We extracted the GPS, and it dose-dependently inhibited the tumorigenicity of hepatocellular carcinoma cells in nude mice. GPS treatment resulted in a significant (P<0.05) dose-dependent increase in the number of apoptotic cells in vivo and a significant (P<0.05) dose-dependent decrease in hepatocellular carcinoma cell proliferation in vitro. GPS modified multiple key enzymes (p-PI3K, p-AKT, and P53) in P53/PI3K/AKT signaling pathways on DNA or protein levels. Taken together, we extracted the GPS successfully and our findings suggest that GPS functions as a tumor suppressor through influencing the P53/PI3K/AKT pathway in the carcinogenesis of hepatocellular carcinoma and may have therapeutic implications for the clinical management of hepatocellular carcinoma patients.