The L-threonine aldolase from Leishmania major was engineered to improve its diastereoselectivity by a CAST/ISM strategy, providing insights into the relationship between the physico-chemical properties of the substrate access path and diastereoselectivity. The steric hindrance, hydrophobic interaction and π–π interaction cooperated to improve the diastereoselectivity of the enzyme, with a diastereomeric excess (de) value reaching 96.3%_syn from 26.8%_syn.