Notch2, a surface marker in cell lines, is used to isolate, identify and localise pancreatic cancer stem-like cells and is a target for therapy of these cells. Sphere formation was induced in Panc-1 and Bxpc-3 pancreatic cancer cell lines, and Notch2+ cells were separated from Bxpc-3 and Panc-1 cell lines by magnetic activated cell sorting (MACS). Expression of stem cell-related markers, OCT4, Nanog and PDX1, were measured by immunofluorescent (IF) staining. Expression of Notch2 was also determined immunohistochemically in pancreatic tissues. Notch2+ cells were transplanted in subcutaneous of mice. AQP1 and AQP5 were also measured by IF in Bxpc-3 cells. The Notch signal pathway inhibitor, Compound E (CE), was used to treat Notch2+Bxpc-3 cells, and their vitalities were subsequently measured by the CCK-8 method. Positive expression of OCT4, Nanog and PDX1 was observed in Notch2+ cells. Notch2+ cells at centroacinar cell (CAC) and terminal ductal locations expressed AQP1 and AQP5. They were strongly tumourigenic in mice, and CE inhibited proliferation of Notch2+ Bxpc-3 cells to some degree. OCT4 and Nanog can be used as markers of self-renewal in pancreatic cancer stem cells. Notch2+ cells in human pancreatic cancer Bxpc-3 and Panc-1 cell lines had the properties of cancer stem cells. The results suggest that Notch2+ pancreatic cancer stem-like cells had a close relationship with CAC.