Abstract: Objective To establish methotrexate(MTX) enantiomers resistant human NSCCL A549 cell line ( L-(+)-MTX/A549,D-(-)-MTX/A549) derived from the A549 cell line and observe its biological characters. Methods A549 cells were exposed to intermittently and progressively increasing doses of MTX enantiomers.Two MTX enantiomers (L and D) resistant human NSCLC A549 cell lines were established.The morphology was observed by inverted phase contrast microscope,the sensitivity of the L-(+)-MTX/A549,D-(-)-MTX/A549 cell lines to MTX enantiomers was measured by cytotoxicity test.The cell growth curve was determined by MTT assay,the change of CFSE fluorescence intensity and cell cycle distribution were analyzed by flow cytometry assay. Results The L-(+)-MTX/A549 resistance index was 6.0 and D-(-)-MTX/A549 resistance index was 20.2.Inverted phase contrast microscope showed morphological changes of L-(+)-MTX/A549 and D-(-)-MTX/A549.Cell growth curve demonstrated that the proliferation abilities of L-(+)-MTX/A549 was decreased significantly,while the cell proliferation rate of D-(-)-MTX/A549 was similar to that of A549.The flow cytometry results showed L-(+)-MTX/A549 and D-(-)-MTX/A549 cells at S phase were reduced (P<0.05) ， with an increased at G₀/G₁ phase (P<0.05),the CFSE mean fluorescence intensity of A549 parent cells,L-(+)-MTX/A549 and D-(-)-MTX/A549 were (6.08±0.55),(7.72±0.30),(6.90±0.18).Two enantiomers resistance cell lines have a clear difference in Chirality. Conclusion We established two MTX enantiomers resistant human A549 cell lines and observed their different biological characteristics.It provides a new experimental model for further studying the mechanism of MTX enantiomers resistance.