In the field of artificial metalloenzyme (ArM) catalysis, how to identify the critical factors affecting the catalytic activity and enantioselectivity remains a challenge. In this work, the mechanism of enantioselective reduction of imine catalyzed by using [Rh(Me4Cpbiot)Cl2]·S112H Sav (denoted as S112H) and [Rh(Me4Cpbiot)Cl2]·K121H Sav (denoted as K121H) was studied by using molecular dynamics (MD) simulations combined with density functional theory (DFT) calculations. Four binding modes of imine, two proton sources (hydronium ion and lysine) and eight proposed reaction pathways were systematically discussed. The results showed that due to the anchoring effect of the mutation site of ArMs, the rhodium complex which oscillated like a pendulum was bound to a specific conformation, which further determined the chirality of the reduced product. C-Hπ, cation-π and ππ weak interactions played an important role in imine binding, and the favorable binding mode of imine was catalyzed by S112H in landscape orientation and catalyzed by K121H in portrait orientation, respectively. LYS121 is the most possible proton source in the S112H catalytic process while the proton source in the K121H catalytic process is the hydronium ion of the active sites. Furthermore, based on the reaction mechanism, modification of Rh(Me4Cpbiot)Cl2 was carried out in S112H and K121H, and the results suggested that the reaction barrier could be effectively reduced by replacing the methyl groups on Cp* with an amino group. This work gives a fundamental understanding of the mechanism of ArMs toward the imine reduction reaction, in the hope of providing a strategy for reasonable designs of ArMs with high enantioselectivity.