The mechanism of histidine-catalyzed asymmetrical aldol reaction of acetone with benzaldehyde was studied by using B3LYP method of density functional theory at the levels of 6-31G(d,p) and cc-pvdz basis sets. The calculation results showed that the reaction mechanism included four steps: (I) nucleophilic attack of histidine on acetone to form alcohol intermediate Inter-A through the transition state TS1 (considered a rate control step because the activation energy (49.95 kcal/mol) was relatively high); (II) dehydration of the alcohol intermediate to form the cis- or trans-enamine through the transition states TS3 and TS4 with the energy barriers of 36.12 and 38.15 kcal/mol; (III) electrophilic addition of cis-enamine or trans-enamine with benzaldehyde to form imine Inter-C or Inter-E through the transition states TS8, TS9, TS10, and TS11 (energy barriers 18.43, 22.34, 13.24, and 13.24 kcal/mol, respectively); (IV) after combination of the imine intermediate with water through the transition states TS12, TS13, TS14, and TS15 (energy barriers 22.79, 34.6, 28.2, 25.12 kcal/mol, respectively), removal of the histidine catalys to obtain the final S or R aldol product. Through analyzing the potential energy profile of reaction, we found that the histidine-catalyzed reaction of acetone with benzaldehyde was more energetically favorable to obtain the R-product (ee value >99%). Solvent effects computed with a polarizable continuum model (PCM) indicated that the DMSO and water can reduce the reaction energy barrier.