AbstractCancer stem cells (CSCs) are a pivotal target for eradicating hepatocellular carcinoma (HCC). We previously reported that distinctive CSCs regulating tumorigenicity (EpCAM⁺ CSCs) and metastasis (CD90⁺ CSCs) have different epithelial/mesenchymal gene expression signatures. Here, we examined the influence of sorafenib, a multiple-receptor tyrosine kinase inhibitor used as a first-line treatment for advanced HCC, on EpCAM⁺ and CD90⁺ CSCs. CD90⁺ cells showed higher c-Kit gene/protein expression than EpCAM⁺ cells. Sorafenib treatment reduced the number of CD90⁺ cells with attenuated c-Kit phosphorylation, whereas it enriched the EpCAM⁺ cell population. We evaluated the role of CD90⁺ and EpCAM⁺ CSCs in vivo by subcutaneously injecting these CSCs together in immune-deficient mice. We observed that sorafenib subtly affected the suppression of primary tumor growth maintained by EpCAM⁺ CSCs, but completely inhibited the lung metastasis mediated by CD90⁺ CSCs. We further evaluated the effect of sorafenib on extracellular vesicle (EV) production and found that sorafenib suppressed the production of EVs containing TGF-β mRNA in CD90⁺ cells and inhibited the cell-cell communication and motility of EpCAM⁺ cells. Our data suggest the following novel effects of sorafenib: suppressing CD90⁺ CSCs and inhibiting the production of EVs regulating distant metastasis.