(D)ensity functional theory has been applied to elucidate the mechanism of the O-demethylation reaction that generates serotonin from 5-methoxytryptamine (5-MT); a process that is efficiently catalyzed by P450 CYP2D6. Two substrates, the neutral 5-MT and the protonated 5-MTH+, were used to probe the reactivity of CYP2D6 compound I. Notably, the H-abstraction process is found to be slightly more facile for 5-MT. However, our DFT augmented by docking results show that the amino acid Glu216 in the active site holds the NH3+ tail of the 5-MTH+ substrate in an upright conformation and thereby controls the regioselectivity of the bond activation. Thus, the substrate protonation serves an important function in maximizing the yield of scrotonin. This finding is in accord with experimental conclusions that 5-MTH+ serves as the substrate for the CYP2D6 enzyme. The study further shows that the H-abstraction follows two-state reactivity (TSR), whereas the rebound path may involve more states due to the appearance of both Fe(IV) and Fe(III) electromers during the reaction of 5-MTH+.