Increasing evidence has revealed the importance of cancer stem cells (CSCs) in carcinogenesis. Although liver CSCs have been identified in hepatocellular carcinoma (HCC) cell lines, no data have shown the presence of these cells in human settings. The present study was designed to delineate CSCs serially from HCC cell lines, human liver cancer specimens to blood samples, using CD90 as a potential marker. The number of CD90⁺ cells increased with the tumorigenicity of HCC cell lines. CD45⁻CD90⁺ cells were detected in all the tumor specimens, but not in the normal, cirrhotic, and parallel nontumorous livers. In addition, CD45⁻CD90⁺ cells were detectable in 90% of blood samples from liver cancer patients, but none in normal subjects or patients with cirrhosis. A significant positive correlation between the number of CD45⁻CD90⁺ cells in the tumor tissues and the number of CD45⁻CD90⁺ cells in the blood samples was identified. CD90⁺ cells sorted from cell lines and CD45⁻CD90⁺ cells from the tumor tissues and blood samples of liver cancer patients generated tumor nodules in immunodeficient mice. Serial transplantation of CD90⁺ cells from tumor xenografts generated tumor nodules in a second and subsequently third batch of immunodeficient mice. Treatment of CD90⁺ CSCs with anti‐human CD44 antibody induced cell apoptosis in a dose‐dependent manner. Conclusion: Identification of CD45⁻CD90⁺ CSCs in both tumor tissues and circulation suggests that CD45⁻CD90⁺ could be used as a marker for human liver cancer and as a target for the diagnosis and therapy of this malignancy. (HEPATOLOGY 2008.)