Lipase-catalyzed oligomerization of alkyl D- and L-lactate monomers (RDLa and RLLa, respectively) was studied for the first time. It has been found that the oligomerization occurs enantioselectively only for D-lactates to give oligomers up to heptamers of lactic acid (LA) in good to high yields by using primary C1 to C8 alkyl groups and sec-butyl group for D-lactate monomers. No reaction happened for all L-lactates in similar conditions. Lipase-catalyzed hydrolysis of alkyl D- and L-lactates was also examined, revealing that the hydrolysis took place for both D- and L-lactates, although L-lactates proceeded a couple of times slower. The hydrolysis results clearly demonstrate that the lipase catalysis mechanism involves an acyl-enzyme intermediate (EM) formation via the acylation step from both D- and L-lactates as a rate-determining step, and the subsequent deacylation step, a nucleophilic attack of water to the EM, takes place to produce free LA. On the other hand, in the oligomerization of D-lactates, the deacylation step, in which a sec-alcohol group of the monomer or of the propagating chain-end attacks to the EM, is only allowed for the sec-D-alcohol group to give a one-LA-unit-elongated oligomer. L-Lactates form the EM; however, the subsequent deacylation reaction with both the sec-L- and sec-D-alcohol groups does not take place, failing in the oligomerization to occur. These results provide with the first direct evidence in the lipase catalysis that the enantioselection is governed by the deacylation step. In the co-oligomerization between L- and D-lactates, the L-isomer retarded the reaction rate of the D-isomer, which was found due to the function of the former as a competitive inhibitor in the acylation step toward the latter.