Serine racemase (SR) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme catalyzing the racemization of S-serine to R-serine, and also oxidizes R-serine to pyruvate; however, the enzymatic racemization reaction is not fully understood. Therefore, quantitative in silico analysis of mammalian SR was performed followed byD-amino acid oxidase (DAO) analysis.The downloaded stereo structures of Rattus norvegicus (PBD ID: 3hmk) and mammalian (PDB ID: 3l6r and 3l6b) SR were optimized using molecular mechanic (MM2) calculation. Furthermore, the stereo structure of mammalian SR was constructed using 3hmk and the amino acid sequence data. The atomic partial charge (apc) of the target atoms was calculated using MOPAC-PM5. The atomic distances, bond angles, and apc of the mutants were used to study the enzyme reaction mechanism.The serine carboxyl group contacted lysine-56, where its amino group contacted the oxygen molecules of PLP aldehyde. The apc of C indicated that S-serine was selectively oxidized. Manganese located at another side of PLP is directly involved in the serine racemization by forming bonding with the hydroxyl group of serine. The enzyme racemization activity was quantitatively related to the bond angle of the substituted serine rather than to that of alanine. However, 316r and 316b lack several amino acid residues; therefore, the selective oxidation of S84 mutants was not clearly demonstrated.