Objective In order to obtain a model of the HEK293 cells infected by porcine endogenous retroviruses PERV, by means of the G418|resistant HEK293 cell line established. Methods The plasmid pIRESneo containing neo gene was purified and transfected into HEK293 cells with Lipofectin. The transfected cells would be survived in the further culture medium containing G418 antibiotic as the neo gene could express a G418 resistant products. The identification of the G418 resistant HEK293 (G418 RHEK293) cells was conducted by polymerase chain reaction with the specific primers of neo gene. Results The G418 RHEK293 cell line was established successfully. There were no differences among G418 RHEK293 cells and normal HEK293 cells morphologically and propagatively. The neo gene DNA fragment could be amplified by PCR with specific primers in the genomic DNA of G418 RHEK293 cells. Conclusion The G418 RHEK293 cells were established with transfection of plasmid pIRESneo by Lipfectin, which may be useful as a cell model for the research of PERV.