(S)-Scoulerine 9-O-methyltransferase (SMT), belonging to the S-adenosyl-L-methionine (SAM)-dependent O-methyltransferase family, is an essential enzyme in the berberine biosynthetic pathways. In order to study the interactions of SMT with its substrate and further to understand the catalytic mechanism and substrate specificity, a three dimensional model of SMT from Coptis chinensis was constructed by homology modeling using the crystal structure of caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) as a template. The three dimensional structure of SMT, which was mainly composed of α-helices and some β-sheets, was similar to that of COMT. In contrast with COMT, the non-conserved residues in the substrate binding pocket of SMT might be responsible for their differences in the substrate specificity. Val119 and Asp254 in SMT were the key residues for orienting substrate for methylation as both residues had H-bonds with (S)-scoulerine. The methylation of (S)-scoulerine involved deprotonation of the 9-hydroxyl group by His253 and Asp254 in SMT followed by a nucleophilic attack on the SAM-methyl resulting in the product, (S)-tetrahydrocolumbamine.