Abstract: Lactate dehydrogenase (LDH) is one of the important enzymes for anaerobic glycolysis and gluconeogenesis. It could catalyze pyruvate to form lactic acid, which had high application value in food fermentation industry. However, LDH was easily affected by high temperature, which leaded to the decline of lactic acid production. To study the effects of different temperature on the conformation and activity of LDH, molecular dynamics simulations at four different temperatures (37, 55, 70 and 85 °C) were performed for 80 ns, respectively. The overall conformational changed and the differences of enzyme active centers were analyzed. The results showed that LDH was relatively stable at 37 and 55 °C, and the root mean square error, root mean square fluctuation, radius gyration and solvent accessible surface area of LDH increased significantly at 70 and 85 °C. Moreover, the secondary structure of protein had changed greatly at 85 °C, which indicated that high temperature would lead to protein conformational instability. Comparing the binding ability of pyruvate at 37 and 85 °C, which was the substrate of the enzyme, we found that high temperature would increase the distance between the residues of pyruvate binding sites, and then destroy the microenvironment of substrate molecular binding. Therefore, the denaturation of LDH occurred when the temperature exceeded 70 °C, and the denaturation degree was positively correlated with the increase of temperature. This would lead to the loss of its enzyme activity, which was not conducive to the application value in food industry. In this study, the effects of four different temperatures on LDH were analyzed at atomic level, and the key information of enzyme activity and conformation changes were revealed, which provided theoretical support for the selection of appropriate temperature in the fermentation process of lactic acid products.