Purpose <p>Hepatocellular carcinoma (HCC), the major subtype of primary liver cancer, is one of the most prevalent and lethal malignancies globally. Limb-bud and heart (LBH), a highly conserved transcriptional cofactor, regulates embryonic development and is implicated in various human diseases, including cancer. However, whether and how LBH relates to liver cancer progression remains elusive.</p> Methods <p>The expression patterns and prognostic value of LBH in HCC were evaluated using data from The Cancer Genome Atlas (TCGA) database. The role of LBH during HCC progression was characterized through in vitro cellular experiments, as well as subcutaneous xenograft and hydrodynamic tail vein injection (HTVi)-induced spontaneous HCC mouse models. Transcriptome sequencing (RNA-seq), quantitative real-time PCR (qPCR), the Cistrome Data database, and chromatin immunoprecipitation sequencing (ChIP-seq) were utilized to explore the signaling pathways governing LBH gene transcription. Mass spectrometry was performed to identify Mortalin as a binding partner of LBH, which was further verified by co-immunoprecipitation (co-IP) and immunofluorescence (IF) assays. Finally, in vitro rescue experiments were conducted to clarify the role of Mortalin in mediating the oncogenic effects of LBH in HCC.</p> Results <p>LBH is significantly upregulated in HCC tissues and functions as an oncogenic driver. The aberrant upregulation of LBH in HCC is attributable to transcriptional regulation by the TGF-β/Smad signaling pathway. LBH notably promotes tumor growth both in vitro and in mouse models. Furthermore, LBH physically interacts with the oncoprotein Mortalin, and the two proteins colocalize in the cytoplasm of HCC cells. Functionally, Mortalin is essential for LBH-mediated oncogenic promotion in HCC.</p> Conclusions <p>Collectively, these findings demonstrate that the TGF-β/LBH/Mortalin axis plays a crucial role in HCC progression, highlighting a potential therapeutic target for the treatment of liver cancer.</p>