Objective:To construct recombinant eukaryote expression vector containing Gal-3 gene and detect protein expression in NIH/3T3 cells. Methods:Gal-3 gene was obtained by PCR from cancer cells. PCR product of Gal-3 was then clone into the eukaryote expression vector pEGFP-N1,then the recombinant vector pEGFP-Gal-3 was transfected into the NIH/3T3 cells. After the transfection of the recombinant vector,the protein expression of Gal-3 in NIH/3T3 cells was detected by Western blot. The effect of Gal-3 on cell proliferation was investigated by MTT assay. Results:The recombinant eukaryote expression vector encoding Gal-3 was constructed restriction enzyme analysis and nucleic acid sequence successfully. The expression of Gal-3 in NIH/3T3 cells transient transfected by recombinant plasmid could be detected by Western blot. In addition,Gal-3 protein could promote the proliferation of NIH/3T3 cells. Conclusion:The new recombinant expression vector pEGFP-Gal-3 was constructed and expressed successfully in NIH/3T3 cells of rats. Furthermore,our study found that Gal-3 protein could promote the proliferation of NIH/3T3 cells.