The Notch signaling pathway plays a role in cell proliferation, differentiation. Emerging data have revealed aberrant Notch3 expression in hepatocellular carcinoma (HCC). However, whether Notch3 plays a role in tumorigenesis or tumor progression is unclear. In this study, we found that over 71.8% of the cases studied had high Notch3 expression levels (n= 32); Notch3 expression positively correlated with alpha-fetoprotein (AFP) levels (p= 0.0311) and negatively correlated with the differentiation grade (p= 0.042). We demonstrated that the patients with higher levels of Notch3 expression commonly had a poor prognosis. We discovered that Notch3 expression is inversely correlated with β-catenin content but positively associated with the protein level of Nanog. In parallel, we found that Notch3 attenuation resulted in the upregulation of β-catenin and the downregulation of Nanog in the hepatoma cell lines QGY7701 and HepG2. The downregulation of Notch3 enhanced the sensitivity to cisplatin in the QGY7701 and HepG2 cells and inhibited the ability of QGY7701 cells to form tumors. The Notch3-positive cells had higher levels of aldehyde dehydrogenase (ALDH) activity, and a tendency to differentiate into Notch3-negative cells. In conclusion, our study demonstrated that Notch3 plays a role in modulating the stemness of tumor cells via the inactivation of the Wnt/β-catenin pathway.