Cancer stem cells (CSCs) are critical players in the pathogenesis of human-associated cancers. It is well established that the stemness of CSCs is modulated by microRNA (miRNA). In the current study, the miR-338-3p deficiency increased self-renewal and tumor malignancy in hepatic CSCs. Nevertheless, miR-338-3p overexpression suppresses tumorigenesis and self-renewal in liver CSCs. Mechanistically, miR-338-3p specifically targets SOX4 in liver CSCs. Moreover, miR-338-3p-associated downregulation of SOX4 prevents tumorigenesis and self-renewal in the CSCs of the liver. The miR-338-3p overexpression in hepatocellular carcinoma (HCC) cells was responsive to lenvatinib-induced apoptosis and cell progression inhibition. Patients' cohort shows that miR-338-3p may predict Lenvatinib benefits in HCC patients. Furthermore, by decreasing the miR-338-3p overexpression sensitivity to Lenvatinib-induced cell death in HCC cells, SOX4 may be a potential therapeutic candidate. In conclusion, miR-338-3p has a considerable function in liver CSC self-renewal and tumor development, making it a promising therapeutic target against HCC.© 2025. The Author(s).