Background <p>Patients with advanced hepatocellular carcinoma (HCC) often develop cholestasis and exhibit poor clinical outcomes. Current evidence suggests that dysregulated bile acid metabolism may contribute to HCC progression, although the underlying molecular mechanisms remain unclear. Among the bile acids, glycochenodeoxycholic acid (GCDCA) is a key component of cholestasis. Under pathological conditions, hepatic progenitor cells (HPCs) transform into fibroblasts or tumor-initiating cells, directly promoting liver fibrosis and HCC development. This study aimed to investigate the regulatory role of GCDCA in the activation and differentiation of HPCs to elucidate its potential mechanisms in hepatocarcinogenesis.</p> Methods <p>Single-cell RNA sequencing was used to infer the heterogeneity of cancer-associated fibroblasts (CAFs) and their differentiation relationship with HPCs in a diethylnitrosamine-induced rat model. The TCGA and GEO datasets were used to assess the prognostic value of inflammatory CAFs (iCAFs) in patients with HCC. Transcriptomic sequencing, functional assays, and in vivo experiments were performed to validate the role of GCDCA in HCC pathogenesis.</p> Results <p>We identified a CAF subset closely associated with the development of HCC (PDGFRA⁺CAFs). GCDCA promotes the differentiation of HPCs into PDGFRA⁺CAFs, which is mediated by sphingosine-1-phosphate receptor 2 (S1PR2). Treatment with the S1PR2 inhibitor JTE-013 markedly inhibited the progression of the pro-fibrotic phenotype in HPCs, which consequently reduced tumor growth and fibrosis in rats.</p> Conclusions <p>GCDCA-induced differentiation of HPCs into PDGFRA⁺CAFs plays a critical role in HCC progression, driven by the S1PR2 receptor. These results provide new insights into the mechanisms underlying HPCs-mediated hepatocarcinogenesis. Targeting S1PR2 represents a promising therapeutic strategy for HCC with potential benefits in reducing fibrosis and tumorigenesis.</p>