Objective:To establish an NIH3T3 cell line that stably expresses GFP-V12Rac1(constitutively active Rac1 fused with a GFP tag). Methods:Plasmids and lentiviral vectors containing GFP-V12Rac1 and GFP were constructed. NIH3T3 was infected with lentiviral vectors,and cells stably expressing genes of interest were selected by flow cytometry. A cell spreading assay was used to confirm that exogenous GFP-V12Rac1 was of normal function;a Boyden chamber assay was used to test the motility of established cell lines. Results:Stable cell lines expressing GFP or GFP-V12Rac1 were established. GFP-V12Rac1 promoted cell spreading. Chemotaxis of established cell lines was confirmed. Conclusion:NIH3T3 cell lines stably expressing GFP-V12Rac1 were successfully established using lentiviral methods;exogenous GFP-V12Rac1 was of normal function;chemotaxis of the cell lines was confirmed. These cell lines can be used as model cells for further study of active Rac1 targeting.