Objective: To construct a lentiviral expression vector for RNA interference (RNAi) of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods: Three pairs of human VIM gene short hairpin RNA (shRNA) sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction (PCR) and DNA sequencing analysis. Real-time PCR and Weste blotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells.The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 cells were validated by real-time PCR and Weste-blotting. Results: An effective Lv-VIM-shRNA was successfully constructed.The titer of lentivirus was determined on 2×109TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion: An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.