Objective To construct the recombinant eukaryon expression plasmid containing human amelogenin (hAm) gene and transfect mammalian cell line NIH3T3 for construction of cells with stable expression of recombinant hAm. Methods hAm gene was inserted into eukaryon expression vector pcDNA3.1/myc-His (-) A with restriction enzyme EcoRⅠ and BamHⅠ, and recombinant plasmid pcDNA3.1/myc-His (-) A-hAm containing hAm gene was confirmed by restriction  endonuclease mapping and sequencing. pcDNA3.1/myc-His (-) A-hAm was transfected into NIH3T3 cells by LipofectamineTM2000, and was selected by G418 for positive cell clones. Cells with stable expression of hAm was constructed, and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results Restriction endonuclease mapping and sequencing revealed that the inserted sequences were accurate in recombinant plasmid pcDNA3.1/myc-His (-)A-hAm. Expression of hAm with molecular weight of 28 000 was detected by SDS-PAGE and Western blotting in NIH3TS cells transfected with recombinant plasmid, which was in line with the prediction. Conclusion The recombinant eukaryon expression system containing hAm has been successfully constructed, and NIH3T3 cells with stable expression of recombinant hAm is obtained.