CD90 has been identified as a marker for liver cancer stem cells (CSCs) that are responsible for tumorigenic activity, but it is not known how CD90(+) cells contribute to tumor initiation and progression. Our data demonstrated that high expression of CD90 in Hepatocellular Carcinoma (HCC) tissues correlated with venous filtration in HCC patients. CD90(+) cells isolated from HCC cell lines exhibited increased tumorigenicity, chemoresistance, tumor invasion and metastasis. Notch pathway was activated in CD90(+) cells and we found that inhibition of Notch pathway in CD90(+) CSCs decreased tumorigenicity, cell invasion, migration and expression of stem cell related genes. Activation of Notch pathway in CD90(-) cells induced self-renewal, invasion and migration. Furthermore, we observed that cancer stem cell features were facilitated by stimulating G1-S transition in the cell cycle phase and inhibiting apoptosis mediated by Notch pathway. Our findings suggested CD90 could be used as a potential biomarker for HCC CSCs, and that cancer stem cell activity was elevated through up activated Notch pathway in CD90(+) CSCs.