〔Abstract〕 Objective To understand how warfarin inhibition is hindered by the vitamin K epoxide reductase(VKORC1) resistant mutations. Methods An electrophoretic mobility assay was conducted based on the principle that binding with warfarin resulted in changes in VKORC1 electrophoretic mobility. The activity of VKORC1 and the half maximal inhibitory concentration(IC50) of warfarin inhibiting VKORC1 were detected by ELISA. These experiments evaluated the binding ability of VKORC1 resistant mutants to warfarin. Results With warfarin bound, VKORC1 was protected from N-ethylmaleimide(NEM) modification and showed increased electrophoretic mobility, which was dependent on an unmodified cysteine on the flexible transmembrane helix 1(TM1) of VKORC1. Increasing the warfarin concentration could shift more VKORC1 towards the species with the fast mobility. Compared to the wildtype VKORC1, the fast mobility fraction became less or disappeared in warfarin-resistant mutants, indicating weakened binding of warfarin. In addition, VKORC1 mutants with the weaker electrophoretic mobility shift indicated the stronger warfarin resistant. Conclusion Weakened warfarin binding is the primary cause of warfarin resistance.