<p>Primary and secondary bile acid (BA) levels are elevated in patients with hepatocellular carcinoma (HCC). BAs are important signaling molecules that regulate CYP8B1 expression by targeting nuclear and membrane receptors. In this study, we aimed to determine the function of CYP8B1 in HCC. Examination of HCC tissue and bioinformatic analysis revealed that CYP8B1 expression is downregulated in HCC tissues and is associated with good prognosis. Cholic acid promoted Huh7 cell proliferation and migration by inhibiting CYP8B1 expression. Both in vitro and in vivo, CYP8B1 inhibited the proliferation, invasion, and migration of HCC cells. Nanopore long-read RNA-sequencing analysis identified PAK4 as a potential target of CYP8B1, and the MAPK pathway was associated with CYP8B1 expression. CYP8B1 inhibited PAK4 expression and Raf/MEK/ERK phosphorylation. Tissue microarray analysis also verified a strong correlation between CYP8B1 and PAK4 expression. In vitro Cell Counting Kit 8 assays and in vivo orthotopic liver tumor model analyses showed that CYP8B1 restores sorafenib sensitivity in resistant HC, suggesting its potential as a therapeutic target. IP-MS of CYP8B1 and transcription factor prediction of PAK4 revealed STAT1 as a potential transcription factor for PAK4, which may directly bind to CYP8B1. Chromatin immunoprecipitation confirmed that u-STAT1 directly binds to the PAK4 promoter, not p-STAT1. Overall, CYP8B1 binds to u-STAT1 in the cytoplasm, reducing the translocation of u-STAT1 from the cytoplasm to the nucleus, thereby inhibiting the transcription of PAK4 and ultimately inhibiting the phosphorylation of Raf/MEK/ERK. Our findings indicate that the CYP8B1/PAK4 axis is important in HCC progression and elucidate the mechanism by which BAs promote HCC. Thus, CYP8B1 is a potential therapeutic target for the clinical treatment of HCC.</p><p></p>