ETHNOPHARMACOLOGICAL RELEVANCE: Alpinia officinarum Hance (A. officinarum), a perennial herb used in the treatment of digestive system cancers, holds significant value for the Li people of Hainan as a traditional Chinese medicine. (R)-5-hydroxy-1,7-diphenyl-3-heptanone (DPHC), a diarylheptanoid component is derived from A. officinarum. Diarylheptanoids have demonstrated anti-proliferative effects on breast cancer cells, neuroblastoma cells, and other tumor cells. However, the pharmacological activity of DPHC in improving hepatocellular carcinoma (HCC) remains undefined. AIM OF THE STUDY: To elucidate the anti-HCC effects of DPHC derived from A. officinarum and explore its underlying mechanistic pathways both in vivo and in vitro. MATERIAL AND METHODS: The effects of DPHC on HCC cell lines were evaluated in vitro using cell counting kit-8, EdU cell proliferation assays, a wound healing assay, a three-dimensional tumor spheroid model, and flow cytometry. The ability of DPHC to ameliorate HCC was assessed in vivo via a nude mouse subcutaneous xenograft tumor model, serum biochemical marker detection, and hematoxylin-eosin staining. The molecular mechanism of DPHC in HCC was elucidated through a combination of transcriptome sequencing, cell transfection, immunohistochemistry assay, immunofluorescence staining, quantitative reverse transcription-PCR, and western blot analysis. RESULTS: DPHC induced significant G0/G1 phase arrest and apoptosis in HepG2 and HCCLM3 cells while also markedly inhibiting tumor growth in nude mice. Mechanically, DPHC directly interacted with centromere-associated protein U (CENPU) to suppress its expression. The reduced expression of CENPU results in decreased interaction with the transcription factor E2F6, thereby affecting the transcriptional activity of the transcription factor E2F1. This subsequently inhibits the expression of downstream cell cycle factors (CCND1, CDK4, and CDK1) and increases apoptosis factors (Caspase 3 and Caspase 9). CONCLUSIONS: DPHC from A. officinarum specifically modulates the function of CENPU in the cell cycle and apoptosis to ameliorate HCC. Our study revealed the anti-HCC effect and underlying mechanism of DPHC, offering new insights and potential targets for HCC treatment.