OBJECTIVE: To investigate the difference in gut microbiota between population with damp-heat constitution (DHC) and balanced constitution (BC). METHODS: A multi-centered cross-sectional case-control study was conducted, which included 249 participants with damp-heat constitution or balanced constitution. Baseline information of participants was collected, and stool samples were collected for gut microbiota analysis. Principal coordinate analysis, linear discriminant analysis effect size analysis, receiver operating characteristic, random forest model, and phylogenetic investigation of communities by reconstruction of unobserved states methods were used to reveal the relationship between gut microbiota and the damp-heat constitution. RESULTS: Compared to those in the BC group, the richness and diversity of the microbiota, specifically those of several short-chain fatty acid producing genera such as Barnesiella, Coprobacter, and Butyricimonas, were significantly decreased in the DHC group. Regarding biological functions, flavonoid biosynthesis, propanoate metabolism, and nucleotide sugar metabolism were suppressed, while arachidonic acid metabolism and glutathione metabolism were enriched in the DHC group. Finally, a classifier based on the microbiota was constructed to discriminate between the DHC and BC populations. CONCLUSION: The gut microbiota of the DHC population exhibits significantly reduced diversity and is closely related to inflammation, metabolic disorders, and liver steatosis, which is consistent with clinical observations, thus serving as a potential diagnostic tool for traditional Chinese medicine constitution discrimination.