Edible mushroom polysaccharides demonstrate a range of bioactivities and have been investigated as potential adjuvant therapies for cancer treatment. Our prior research found Lactariks hatsudake Tanaka polysaccharide (LHP) effective against hepatocellular carcinoma, but the mechanisms remain unclear. This study aims to clarify the anti-cancer molecular mechanisms by examining the effects on specific signaling pathways through in vivo and in vitro analyses. Animal studies showed that LHP significantly inhibited the growth of transplanted liver cancer without toxicity. Histological and immunohistochemical analyses revealed that LHP increased nuclear cohesion in tumor cells and altered the expression of key regulatory proteins such as protein kinase B (Akt), nuclear factor κB (NF- κB), caspase-3, p21, and p53. These findings suggest that LHP’s anticancer effects involve promoting apoptosis and disrupting the cell cycle. At the cellular level, assays such as 3-(4,5- dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS), flow cytometry, and transwell invasion confirmed that LHP inhibited cancer cell proliferation and induced apoptosis. Polymerase chain reaction and Western blot assays revealed that LHP intervention reduced cyclin-dependent kinase 4 activity by upregulating p53 and p21, leading to cell cycle arrest and inhibited cell proliferation. LHP also promoted apoptosis by altering Bcl-2-associated X protein, B-cell lymphoma 2, and cleaved caspase-3 levels. Moreover, LHP could affect the Akt, NF-κB, and mitogen-activated protein kinase signaling pathways to exert anticancer effects. These findings underscore the potential of LHP as a novel and multifaceted therapeutic agent. Furthermore, the elucidation of LHP-related mechanisms offers a crucial theoretical foundation for developing innovative and effective treatments for hepatocellular carcinoma. Additionally, this supports its potential development as a functional food or adjuvant therapy for cancer, which holds significant implications and application prospects in both the food and medical fields.