This study characterized cancer stem cells (CSCs) in hepatocellular carcinoma (HCC) cell lines, tumor specimens, and blood samples. The CD90⁺ cells, but not the CD90⁻ cells, from HCC cell lines displayed tumorigenic capacity. All the tumor specimens and 91.6% of blood samples from liver cancer patients bore the CD45⁻CD90⁺ population, which could generate tumor nodules in immunodeficient mice. The CD90⁺CD44⁺ cells demonstrated a more aggressive phenotype than the CD90⁺CD44⁻ counterpart and formed metastatic lesions in the lung of immunodeficient mice. CD44 blockade prevented the formation of local and metastatic tumor nodules by the CD90⁺ cells. Differential gene expression profiles were identified in the CD45⁻CD90⁺ and CD45⁻CD90⁻ cells isolated from tissue and blood samples from liver cancer patients and controls.