At present, classic therapies provide limited benefits to the survival of patients with pancreatic cancer. However, clinically available gene therapy strategies have not been well established. This study investigates the effect of shRNA-mediated inhibition of XIAP and survivin expression on the proliferation, apoptosis, and chemosensitivity of pancreatic cancer cells. Stable inhibition of XIAP and survivin expression in SW1990 and Panc-1 pancreatic cancer cells was established by lentivirus-carried shRNAs. The mRNA and protein expression of XIAP and survivin were detected by real-time PCR and Western blot, respectively. Cell proliferation was measured by MTT assay, and apoptosis was detected by caspase-3/7 activity and Hoechst33342 staining. The lentivirus-carried shRNA significantly inhibited XIAP and survivin expression. Simultaneous inhibition of XIAP and survivin expression in pancreatic cells significantly reduced cell proliferation, increased caspase-3/7 activity, and increased cell sensitization to 5-FU and gemcitabine treatments compared to inhibition of XIAP or survivin expression alone. However, simultaneous silencing of XIAP and survivin showed no significant difference in inducing cell apoptosis compared to silencing of XIAP or survivin expression alone. Simultaneous inhibition of XIAP and survivin expression may be an effective strategy for gene therapy of pancreatic cancer.