Abstract: In this study, L-lactate dehydrogenase (Lr-L-LDH) from Lactobacillus rhamnosus was investigated. The well-known Lr-L-LDH1 was taken as the control, differences between Lr-L-LDH1 and Lr-L-LDH2 annotated from the genome of L. rhamnosus were analyzed. The primary structure, basic properties, hydrophobicity, secondary structure of Lr-L-LDH1 and Lr-L-LDH2 were predicted and analyzed using online websites and professional software. Homology modeling of the tertiary structure, molecular docking of enzymes and substrates, phylogenetic analysis, in vitro cloning, expression, and enzyme activity assays were further studied. The results showed that, in comparison to Lr-L-LDH1, although Lr-L-LHD2 had similar molecular characteristics, secondary and tertiary structures, Lr-L-LHD2 exhibited differences: A shorter sequences, low amino acid identities (48.08%), and a different phylogenetic status. Lr-L-LHD2 also contained a catalytically active site, a highly conserved NAD⁺ binding site sequence (GXGXXG), a three-dimensional active pocket domain, was an NAD⁺ dependent tetrameric L-lactate dehydrogenase, and be activated by fructose 1,6-diphosphate in the cytoplasm for catalyzing the reduction of pyruvate to L-lactic acid. In vitro enzyme activity was significantly lower than Lr-L-LDH1 (P<0.01). In conclusion, both Lr-L-LDH1 and Lr-L-LHD2 showed enzyme activities of L-lactate dehydrogenase, indicating they may regulate the L-lactate metabolism pathway together. Considerations of both Lr-L-LDH1 and Lr-L-LHD2 should be taken for the future molecular modifications of L-LDH in L. rhamnosus, and the study provided the molecular and scientific basis for the gene engineering modifications of lactic acid fermentation industry.