Background: Hypoxic preconditioning may be a key influence on functions of endothelial progenitor cells (EPCs). Material/Methods: To investigate the role and mechanism of the Notch-Jagged1 pathway on endothelial progenitor cells in hypoxic preconditioning, endothelial progenitor cells were randomly allocated into 5 groups: 1 Normoxic control group; 2 Hypoxic blank group; 3 Hypoxic+25 mu M DAPT group; 4 Hypoxic+50 mu M DAPT group; 5 Hypoxic+100 mu M DAPT group. After reoxygenation, protein and mRNA levels of Jagged1 were measured by Western blot and quantitative RT-PCR. The MTT test was used to assess proliferation. ELISA was used to measure NO and VEGF secretion. Results: Hypoxic preconditioning treatment significantly upregulated both protein and mRNA levels of Jagged1 in endothelial progenitor cells. It also enhanced proliferation ability and elevated secretion of NO and VEGF. Furthermore, after blocking the Notch pathway by using DAPT, Jagged1 expression and EP proliferation, migration, and secretion of NO and VEGF were decreased in a dose-dependent manner. Conclusions: Our results suggest the Notch-Jagged1 pathway enhances EPCs proliferation and secretion ability during hypoxic preconditioning.