Objective: To study the effect of 14-3-3σ gene overexpression on the invasive ability of pancreatic cancer PANC-1 cells. Methods: The coding sequence of 14-3-3σ gene was amplified by PCR using human genomic cDNA as a template and inserted into the eukaryotic expression vector pEGFP-N1 to construct the recombinant pEGFP-14-3-3σ plasmid. After identification by restriction endonuclease digestion and nucleotide sequencing, the constructed plasmids were firstly transfected into the HEK293T cells to assess the transfection efficiency, following which they were transfected into the pancreatic cancer PANC-1 cells mediated by liposome and subsequently, the mRNA and protein expression of the target gene in the PANC-1 cells and invasive ability of these cells were detected by real time fluorescence quantitative PCR, Western blot analysis and Transwell invasion assay, respectively. The PANC-1 cells transfected with empty plasmid or without transfection were used as negative and blank control, respectively. Results: Restriction enzyme digestion and DNA sequencing demonstrated that 14-3-3σ gene was correctly inserted into the pEGFP-N1 vector, and the transfection efficiency of pEGFP-14-3-3σ for HEK293T cells reached 65%. Both the 14-3-3σ mRNA and protein expression were increased significantly in the PANC-1 cells after transfection with pEGFP-14-3-3σ. The results of Transwell invasion assay showed the number of the pEGFP-14-3-3σ transfected PANC-1 cells that invaded through the membrane was significantly higher than that of the PANC-1 cells transfected with empty plasmid or without transfection (129.4±19.6 vs 76.4±17.7, 78.7±16.7) (both P<0.05). Conclusion: Overexpression of 14-3-3σ gene can enhance the invasive ability of pancreatic cancer PANC-1 cells.