To establish a pig kidney cell line LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PK1 cells via liposomes.  After selecting with G418, population doubling time, flow cytometry and Western blotting analysis were used to evaluate the cell line.  MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin.  Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst 33342 by BCRP, furthermore, the BCRP’s inhibitor GF120918 was applied to reverse the efflux of Hoechst 33342.  The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell.  The population doubling time of LLC-PK1/BCRP cell was a little longer than that of the parental cell LLC-PK1.  The resistance indexes to mitoxantrone and doxorubicin were 51.95 and 6.09 times, respectively, higher than LLC-PK1 cell.  The efflux of Hoechst 33342 was significantly enhanced and could be reversed by GF120918.  So a LLC-PK1/BCRP cell line was established, which highly expressed BCRP protein successfully.  This cell line could be a valuable model to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.