Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into D-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable pi-pi stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the k(cat)/K-m value of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms similar to 13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.