Objective: To investigate the inhibitory effect of lentiviral vector-mediated RNA interference (RNAi) on XIAP (X-linked inhibitor of apoptosis protein) and survivin expression in human pancreatic cells and its effect on the proliferation, apoptosis and chemosensitivity of pancreatic cells. Methods: The pGCSIL-PUR and pGCSIL-NEO were used to construct the lentiviral vectors containing shRNA targeting XIAP and survivin gene, respectively, and the human pancreatic cancer SW1990 and Panc-1 cells were transfected with these lentiviruses. The cells were screened by puromycin and neomycin double selection and expanded to obtain the stably transfected cell lines. The XIAP and survivin expressions were determined by real-time PCR and Western blot, respectively. The proliferation and chemosensitivity of the cells were detected by MTT assay, and caspase-3/7 activity measurement and DAPI-staining were performed to assess cell apoptosis. Results: The stable SW1990 and Panc-1 cells with inhibition of XIAP and survivin expression were obtained after the effective interference targets of XIAP and survivin screening. MTT results showed that the proliferative abilities of both types of pancreatic cells significantly decreased after the suppression of XIAP or survivin expression, and the combination inhibition of the two genes exerted a strengthened effect. Although XIAP or survivin inhibition had no obvious effect on apoptosis of both types of pancreatic cells, they enhanced the sensitivities of both types of cells to the chemotherapy agents (5-FU and gemcitabine), and this effect was also strengthened by their combination. Conclusion: The lentiviral vector-mediated RNAi targeting XIAP and survivin can effectively inhibit XIAP and survivin expression, and also inhibit proliferation and enhance chemosensitivity of pancreatic cells. The combination inhibition of both genes can exert a synergistic effect.