Abstract: Objective: To investigate the differential expression of enolase in the membrane and cytoplasm of Gardnerella vaginalis and Lactobacillus strains, and to compare the differences in their glycolytic abilities. Methods: Gardnerella strains were isolated, purified, and identified from vaginal secretions of patients with bacterial vaginosis. Lactobacillus strains were isolated from vaginal secretions of healthy women and cultured in vitro for a period of time under the same conditions. The residual concentrations of glucose, lactate, pyruvate, and adenosine triphosphate (ATP) in the bacterial solution were detected. The membrane and cytoplasmic proteins of Gardnerella and Lactobacillus were extracted respectively, and the enolase levels in the cytoplasm and membrane were detected by western blotting. Results: The residual glucose content in the culture medium of Gardnerella was decreased significantly, while the residual glucose content in the culture medium of Lactobacillus was relatively higher, and there was significant difference between the two groups (P＜0.05); the amount of ATP produced by Gardnerella was significantly higher than that of Lactobacillus (P＜0.05). When enolase antibodies were added to the reaction system, the amount of residual glucose was increased, while the amount of ATP, lactate, and pyruvate was decreased. Enolase was present in the cytoplasm of all Gardnerella and Lactobacillus, and the content of enolase in Gardnerella was significantly higher than that in Lactobacillus (P＜0.05). Only some Gardnerella and Lactobacillus strains had enolase on their cell membranes, and the content of enolase on Gardnerella cell membranes was significantly higher than that on Lactobacillus cell membranes (P＜0.05). Conclusion: There is a certain difference in the expression of enolase between Gardnerella vaginalis and Lactobacillus, which to some extent affects the metabolic ability of the two strains.