Abstract: Objective To clone human epidermal growth factor receptor (HER2) gene and establish a MCF-7 cell line with stable co-expression of HER2 and estrogen receptor (ER). Methods The full-length HER2 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line SKBR-3 which over-expressed HER2 protein. The gene fragment was then inserted into the vector pcDNA3.1 to construct the recombinant expression plasmid pcDNA3.1- HER2, which was identified by sequence analysis and transfected into ER-expressing MCF-7 cells via lipofectamine. The positive cell clones were obtained after G418 selection. The MCF-7 cells transfected with HER2-cDNA was assayed by immunocytochemistry, flow cytometry and Western blotting. Results DNA sequencing results showed that HER2 gene was exactly consistent with the sequence reported in GenBank. The MCF-7 cells tansfected with HER2 could highly express HER2 protein as shown by immunocytochemistry, flow cytometry and Western blotting. Conclusion HER2 gene has been cloned successfully and the MCF-7 cell line co-expressing HER2 and ER is obtained.