In hepatocellular carcinoma (HCC), the poor response to the chemotherapeutic agents is partially attributed to the chemoresistance property of cancer stem cells (CSCs). NOTCH signaling pathway plays a crucial role in the chemoresistance through the maintenance of the CSCs. We observed that the NOTCH pathway was activated in HCC CD133(+) cells treated with vincristine (VIN)1 and 5-fluorouracil (5-FU)(2). Therefore, we examined whether inhibition of the NOTCH can improve sensitization of HCC CD133(+) cells to VIN and 5-FU. The Huh7 cell line was pre-incubated gamma-secretase DAPT, as a NOTCH inhibitor, and then treated with IC50 dose of VIN or 5-FU. The CD133(+) cells were then isolated and analyzed for the cell viability, apoptosis, migration and spheroid formation capacities, and gene and protein expression. It was observed that pre-incubation with DAPT significantly downregulated the expression of NOTCH-related genes and led to a significant reduction in VIN- and 5-FU-CD133(+) population. In addition, DAPT pre-incubated VIN- and 5-FU-treated-CD133(+) cells formed fewer spheroids in 3D culture and had a lesser migration capacity in 2D culture. Importantly, DAPT enhanced the apoptosis rate of VIN- and 5-FU-treated CD133(+) cells for 3- and 2-fold, which was correlated with the enhanced expression of proapoptotic BBC3 (BCL-2-binding component 3) and decreased expression of HES1 that was reported to regulate BBC3 negatively. Collectively, it was observed that NOTCH inhibition sensitized the HCC CD133(+) cells to VIN and 5-FU through enhancing BBC3-mediated apoptosis. The results highlighted the role of NOTCH/HES1/BBC3 axis in resistance of CD133(+) cells to VIN and 5-FU. Understanding the molecular mechanisms underlying chemoresistance in HCC CD133(+) cells may help in designing the novel targeted therapies to chemosensitize them. (C) 2020 Elsevier Inc. All rights reserved.