Objective: To construct the recombinant eukaryote expression vector containing Arf6-T27N gene and detect the protein expression in its target cell line,MDA-MB-231. Methods: The fragment of Arf6-T27N gene from expression vector pXS-Arf6-T27N was cloned into the eukaryote expression vector pEGFP-N1,then the recombinant vector pEGFP-N1/Arf6-T27N were transfected into the MDA-MB-231 cells. After transfection of the recombinant vector,the protein expression of Arf6-T27N in MDA-MB-231 cells were detected by Western blot. The effect of Arf6-T27N on cell migration was investigated by Boyden chamber assay. Results: The recombinant eukaryote expression vector carrying Arf6-T27N was constructed successfully. The expression of Arf6-T27N in MDA-MB-231 cells could be detected by Western blot. In addition,Arf6-T27N protein could inhibit the migration of MDA-MB-231 cells. Conclusion: The new recombinant expression vector pEGFP-N1/Arf6-T27N was constructed successfully and the MDA-MB-231 cells which could express Arf6-T27N stably were also successfully made. Furthermore,our study suggested that Arf6-T27N protein could inhibit the migration of MDA-MB-231 cells.