[Objective] This study is to investigate the effect of lactate dehydrogenases on the optical purity of L-lactic acid produced in Bacillus sp. P38. [Methods] Genome annotation result shows that there are three enzymes responsible for lactic acid production: L-lactate dehydrogenase (L-LDH) (encoded by ldhL), D-LDH (encoded by ldhD), and one possible malate/lactate-LDH (M/L-LDH) (encoded by ldhM/L). These enzymes were investigated both in vivo and in vitro to study the relationship between enzymatic activities, gene transcriptions and the optical purity of lactic acid. [Results] M/L-LDH was found mainly to act as L-LDH. The L-LDH catalytic efficiency toward pyruvate was 2.9-fold higher than that of D-LDH and 4.3-fold higher than that of M/L-LDH. The D-LDH activity was not detectable in Bacillus sp. P38 under active staining. The relative transcription levels of ldhL in Bacillus sp. strain P38 were much higher than those of ldhD and ldhM/L at different growth phases, and the transcription ratio of ldhL to ldhD increased from the logarithmic phase to decline phase. [Conclusion] L-LDH is the key enzyme for high optical purity of L-lactic acid produced by Bacillus sp. P38.