The methyl-transfer reaction mechanism catalyzed by Isoflavone O-methyl-transferase (IOMT) and the roles of several residues around the active site are investigated by employing density functional method. The calculations confirm that the proton transfer from daidzein to His257 occurs barrierlessly, and the methyl group is transferred from S-adenosyl-l-methionine (SAM) to phenolate ion in a single SN2 step with a barrier of 17.0 kcal/mol, consistent with experimental value. Glu318 and Asp288 play important roles in lowering the reaction barrier.Graphical abstractView high quality image (45K)Research highlights► Methyl-transfer mechanism catalyzed by IOMT was investigated by density functional method. ► Catalytic roles of some residues in the active site were studied by several quantum chemical models. ► Calculated activation barrier is in good agreement with the experimental data.