In this study, we developed a versatile strategy for biocatalytic resolution of racemates to produce enantiopure α-hydroxyphosphonates. Under mild conditions, a series of racemic α-hydroxyphosphonates were acetylized to S-acetates in approximately 50% yields and >99% ee. The reactions were catalyzed using lipase B from Candida antarctica (CALB), which is commercially available, and conditions for the reactions involving the enzyme have been established. The catalytic efficiency and enantioselectivity of CALB depend on the α-carbon substituents. The enzyme-substrate binding mode was established and the high enantioselectivity of CALB was revealed through docking and molecular dynamics (MD) simulations.