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AIM: To investigate RNA interference targeting signal transducer and activator of transcription-3 (STAT3) on invasion of human pancreatic cancer cells. METHODS: We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV (lentivirus)-STAT3siRNA (STAT3 small interfering RNA) the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP-2) mRNA and protein expression were examined by quantitative PCR and west blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay. RESULTS: We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells. CONCLUSION: The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.