Cytochrome P450 8B1 (CYP8B1) is a critical enzyme in bile acid metabolism. Using multiple databases, including Gene Expression Omnibus, UALCAN (The University of Alabama at Birmingham Cancer data analysis Portal, GEPIA (Gene Expression Profiling Interactive Analysis), TCGA (The Cancer Genome Atlas) and GTEx (Genotype‑Tissue Expression), the present study analyzed CYP8B1 expression and its prognostic value in hepatocellular carcinoma (HCC). The results showed that CYP8B1 expression was significantly lower in HCC compared with normal tissues, and reduced CYP8B1 expression was associated with poor prognosis in patients with HCC. CYP8B1 was overexpressed in HCC cell lines (Huh7 and Hep3b cells); cell proliferation was assessed using Cell Counting Kit‑8 and EdU assays, while apoptosis was evaluated using the TUNEL assay. CYP8B1 overexpression inhibited proliferation and promoted apoptosis in HCC cells. Additionally, analyses via UALCAN and the Metascape platform showed that CYP8B1 expression was negatively associated with YWHAZ (Tyrosine 3/tryptophan 5 monooxygenase activation protein ζ), the regulation of PLK (Polo‑like kinase) activity during G2/M transition, and the intrinsic apoptosis pathway. Immunoblotting revealed that CYP8B1 overexpression decreased YWHAZ levels. Consistently, the expression of cyclin‑dependent kinase 1) and CCNB1 (Cyclin B1), key markers of G2/M transition, was also diminished following CYP8B1 overexpression. Furthermore, the pro‑apoptotic protein Bax was upregulated, while the anti‑apoptotic protein Bcl‑2 was downregulated. In conclusion, CYP8B1 holds promise as a potential prognostic target for HCC.