Objective In order to obtain a feeder layer for ES cells transfected by pTRE-Ins-human gene by means of developmed of the hygromycin-resistant 3T3 cell line. Methods The plasmid pHyg containing hygromycin gene was purified and transfected into 3T3 cells with Lipofectin.The transfected cells would be survived in further culture medium containing hygromycin B antibiotic as the hygromycin gene could express a hygromycin resistant products.The identification of the hygromycin resistant 3T3 cell line was conducted by polymerase chain reaction with the specific primers of hygromycin gene. Results The hygromycin R 3T3 cell line was established successfully.There were no differences among hygromycin R 3T3 cells and normal 3T3 cells morphologically and propagatively. The hyg gene DNA fragment could be amplified by PCR with specific primers in the genomic DNA of hygromycin R 3T3 cells. Conclusion The hygromycin R 3T3 cells were established with transfection of plasmid pHyg by Lipfectin,which may be useful for selecting ES cells transfected by pTRE-Ins-human gene.