Background <p>Many cases of advanced hepatocellular carcinoma (HCC) are resistant to the widely used drug sorafenib, which worsens prognosis. While many studies have explored how acquired resistance emerges during drug exposure, the mechanism underlying primary resistance before treatment still remain elusive.</p> Methods <p>Single-cell lineage tracing and RNA sequencing were performed to identify primary sorafenib-resistant lineages in HCC. Differential gene expression analysis was employed to identify the biomarkers of drug-resistant lineage cells. Cell viability and colony formation assays were adopted to assess the involvement of S100A14 on sorafenib resistance. Co-immunoprecipitation (CO-IP) and mass spectrometry analysis were conducted to uncover the downstream targets and regulatory mechanisms of S100A14 in primary resistance to sorafenib. In vivo mouse xenograft experiments were carried out to assess the effect of S100A14 or its interacting protein glutaminase (GLS) on primary resistance to sorafenib in HCC.</p> Results <p>Single-cell lineage tracing identified a cluster of sorafenib primary resistant cells, and S100A14, a Ca²⁺-binding protein, was determined to be a critical biomarker for primary resistance to sorafenib. Knockdown of S100A14 significantly increases sorafenib treatment sensitivity in HCC cells. Mechanistically, S100A14 binds to GLS and blocks its phosphorylation at residues Y308 and S314, which in turn inhibits its ubiquitination and subsequent degradation. By stabilizing GLS, S100A14 reduces oxidative stress in HCC cells, thereby antagonizing sorafenib-induced apoptosis. Inhibiting S100A14 or GLS significantly improved sorafenib efficacy against xenograft tumors in vivo.</p> Conclusions <p>Our results demonstrate that S100A14 plays a pivotal role in promoting primary resistance to sorafenib by stabilizing GLS to reduce oxidative stress, and acts as a potential therapeutic target to enhance the efficacy of sorafenib in HCC patients.</p> Graphical Abstract <p></p>