An enantiopure (2R,5R)-1,3-oxathiolane was obtained using a multienzymatic cascade protocol. By employing a combination of surfactant-treated subtilisin Carlsberg and Candida antarctica lipase B, the absolute configuration of the resulting 1,3-oxathiolane ring was efficiently controlled, resulting in an excellent enantiomeric excess (> 99%). This enantiopure 1,3-oxathiolane derivative is a key precursor to anti-HIV agents, such as lamivudine, through subsequent N-glycosylation.