Objective： To investigate the effect of downregulation of Notch1 by Notch1 small interfering RNA （siRNA） on chemosensitivity to gemcitabine in pancreatic cancer cells and its mechanism. Methods： Notch1 siRNA was transfected to pancreatic cancer cell lines AsPC1， BxPC3， MIAPaCa2 and Panc1. The transfected pancreatic cancer cells were treated with 10 μmol/L gemcitabine in vitro. The relative quantity of Notch1 mRNA of pancreatic cancer cells was detected by realtime PCR. The inhibition rates of gemcitabinetreated cells were evaluated by CCK8 method. The expression of Bax protein was examined by Western blot， and the caspase 3 activity was detected by CaspACETM assay system kit. Results： The relative quantity of Notch1 mRNA was the highest in BxPC3 cell line and the lowest in Panc1 cells. The inhibition rates of gemcitabine treatedcells were significantly higher in Notch1 siRNA transfection groups than in corresponding siRNA control groups （AsPC1: 675±67 vs 475±68; BxPC3: 905±44 vs 702±42; MIAPaCa2: 809±57 vs 581±60; Ps<005）， with the overexpression of protein Bax. The activity of caspase 3 was also significantly increased in Notch1 siRNA transfection groups compared with corresponding siRNA control groups （AsPC1: 2890±270 vs 1282±344; BxPC3: 5987±677 vs 2727±1188; MIAPaCa2: 2934±406 vs 1459±425; P<005）. Conclusion： Inhibition of Notch signaling pathway by Notch1 siRNA can enhance chemosensitivity to gemcitabine in pancreatic cancer cells through activating apoptosis activity．