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Hepatocellular carcinoma (HCC) is a lethal malignancy requiring novel therapeutic interventions. While Polyporus umbellatus exhibits anti-tumor properties, its specific bioactive pharmacophores and molecular mechanisms remain elusive. This study integrated network pharmacology, computational simulation, and experimental validation to decipher the anti-HCC efficacy of Polyporus umbellatus. Screening identified 11 bioactive sterols, with intersection analysis revealing 63 core targets. Clinical data stratified Checkpoint Kinase 1 (CHEK1) as a critical high-risk oncogene associated with poor prognosis. Molecular dynamics simulations (100 ns) demonstrated that polyporusterone E, a key constituent, forms a thermodynamically stable complex with CHEK1 via high-affinity hydrogen bonding. In vitro assays in HepG2 and HuH-7 cells confirmed that CHEK1 overexpression drives proliferation and metastasis, while its silencing reverses these phenotypes. Crucially, treatment with Polyporus umbellatus extract and purified polyporusterone E significantly compromised HCC cell viability and downregulated CHEK1 expression at transcriptional and translational levels. These findings suggest that polyporusterone E may downregulate CHEK1 expression and modulate CHEK1-associated signaling in HCC cells, providing preliminary evidence for the molecular basis of Polyporus umbellatus and highlighting its potential as a complementary therapeutic strategy for HCC management.