Density functional theory calculations have been performed to gain insights into the catalytic mechanism of the N-quaternized pyridoxal (i.e., 1a)-mediated biomimetic asymmetric Mannich reaction of tert-butyl glycinate 3with N-diphenylphosphinyl imine 2ato give the diamino acid ester 4ain high yield with excellent enantiomeric and diastereomeric selectivity (Science2018, 360, 1438). The study reveals that the whole catalysis can be characterized via three stages: (i) the catalyst 1areacts with the tert-butyl glycinate 3to generate the active carbanion complex IM3. (ii) IM3then reacts with the N-diphenylphosphinyl imine 2agiving the imine intermediate IM8. (iii) IM8undergoes hydrolysis to give the final product anti-4aand regenerate the catalyst 1afor the next catalytic cycle. Each stage is kinetically and thermodynamically feasible for experimental realization. The hydrolysis step in the stage III is predicted to be the rate-determining step during the whole catalytic cycle. Furthermore, the origins of the enantioselectivity and diastereoselectivity for the target reaction, as well as the deactivation of the catalyst 1b, are also discussed.