Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with a dismal prognosis that is largely attributed to the capacity of liver cancer stem cells (LCSCs) to self-renew in response to conventional therapies. Therefore, it is crucial to develop new therapeutic strategies that target LCSCs to improve the clinical outcomes of patients with HCC. We surveyed and analyzed publicly available single-cell TCGA (the cancer genome atlas), single-cell (scRNA-seq) and spatial RNA-sequencing databases from HCC patient specimens for genes uniquely expressed in LCSCs. We generated and characterized LCSCs from patient-derived HCC cell lines and used them as tools to uncover the previously unknown molecular mechanisms associated with the stemness of LCSCs. We selectively screened a bank of natural compounds to identify drugs that can specifically target LCSCs for HCC treatment and documented their effects both and . TCGA analyses showed that SETD1B expression was aberrantly elevated in HCC, correlating with poor prognosis and a distinct molecular signature of stemness. We demonstrated that SETD1B, driven by MAZ, enhances stem characteristics by promoting anchorage-independence, cellular adhesion, tumor sphere formation, and growth via the surface glycoprotein CD24. We identified triptolide (Trip), which serves as a potent suppressor of LCSC stemness by targeting SETD1B for degradation, thereby dramatically attenuating HCC progression and . These findings establish the MAZ/SETD1B/CD24 signaling cascade as a critical regulatory mechanism of LCSC stemness and highlight Trip as a potential therapeutic agent for HCC.