O-Methylation of N-acetylserotonin (NAS) has been identified as the bottleneck in melatonin biosynthesis pathway. In the present paper, caffeic acid O-methyltransferase from Arabidopsis thaliana (AtCOMT) was engineered by rational design to improve its catalytic efficiency in conversion of NAS to melatonin. Based on the notable difference in the terminal structure of caffeic acid and NAS, mutants were designed to strengthen the interactions between the substrate binding pocket of the enzyme and the terminal structure of the unnatural substrate NAS. The final triple mutant (C296F-Q310L-V314T) showed 9.5-fold activity improvement in O-methylation of NAS. Molecular dynamics simulations and binding free energy analysis attributed the increased activity to the higher affinity between the substrate terminal structure and AtCOMT, resulting from the introduction of N-H center dot center dot center dot pi interaction by Phe296 substitution, hydrophobic interaction by Thr314 substitution and elimination of electrostatic repulsion by substitution of Gln310 with Leu310. This work provides hints for O-methyltransferase engineering and meanwhile lays foundation for biotechnological production of melatonin.