Abstract: Objective To construct a novel eukaryotic expression plasmid for recombinant immunotoxin DT390-Rantes and perform preliminary analysis of its function. Methods The gene fragment coding for Rantes was obtained from the liver tissues of C57BL/6 mice using RT-PCR, and inserted into the eukaryotic expression plasmid SRα containing DT390 gene to construct the recombinant plasmid DT390-Rantes-SRα, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinance plasmid DT390-Rantes-SRα was transfected into NIH3T3 cells and its expression was observed by immunofluorescence detection. The activity of the expressed DT390-Rantes in vitro was evaluated by MTT assay. Results The gene fragment of Rantes was correctly inserted into the eukaryotic expression plasmid SRα as verified by restriction endonuclease digestion and DNA sequencing, and could be expressed in NIH3T3 cells. MTT assay confirmed that the expression product DT390-Rantes could kill activated T cells in vitro. Conclusions The recombinant eukaryotic expression plasmid DT390-Rantes-SRα is successfully constructed and expressed in eukaryotic cells. The expressed product can specifically kill activated T cells in vitro.