Metastatic hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. However, the cell population responsible for its metastasis remains largely unknown. Here, we reported that CD133(+)CD44(+/high) defined a subgroup of tumor cells that was responsible for hematogenous metastasis of liver cancers. Immunohistochemical investigation of human HCC specimens revealed that the number of CD133(+) and CD44(+) HCC cells was increased and was associated with portal vein invasion. Purified CD133(+) or CD44(high) HCC cells were superior in clonogenic growth and vascular invasion, respectively. Thus, the combination of CD133 and CD44 was used to define a novel HCC sub-population. CD133(+)CD44(high), but not CD133(+)CD44(low/-), CD133(-)CD44(high) or CD133(-)CD44(low/-) xenografts, produced intrahepatic or lung metastasis in nude mice. Further analysis of human HCC samples by flow cytometry showed that the number of CD133(+)CD44(+) tumor cells was associated with portal vein metastasis. The cDNA microarray analysis of CD133(+)CD44(+) and CD133(+)CD44(-) tumor cells isolated from metastatic HCC patients revealed that these cells comprised of two different populations possessing distinct gene expression profiles. Our results suggest that CD133(+)CD44(+) tumor cells are a particular population responsible for hematogenous metastasis in liver cancers and that these cells might be targets for treatment of HCC metastasis.