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The purpose of this study was to examine whether enterotypes in the gut microbiome could be determined by clustering validity indexes and whether these enterotypes were associated with individual differences in temperament traits.Sixty healthy Korean participants (21 men; 27.5 [8.1] years, 39 women; 34.5 [14.3] years) were asked to answer 60 temperament questions (novelty seeking, harm avoidance, reward dependence, and persistence) from the Korean version of the Temperament and Character Inventory-125. The participants' stool samples were submitted, and relative operational taxonomic units were calculated using 16S rRNA gene-based microarrays. Differences between sexes and age-related effects on the temperament and operational taxonomic unit abundances were determined, and optimal clustering numbers related to enterotypes were examined using connectivity and silhouette width (SW) indexes. Finally, the differences in temperament between enterotypes were examined.There was no significant effect of sex or age on gut microbiota and temperament scores. The hierarchical dendrogram, connectivity, and SW analysis indicated bimodal enterotypes. Bacteroidaceae were more abundant in enterotype 1 (46.24%, N = 45), whereas Prevotellaceae were more abundant in enterotype 2 (43.83%, n = 15). Among the four temperament dimensions, novelty seeking and reward dependence scores were higher in enterotype 1 (10.82 [2.99] and 8.07 [2.51] points) than in enterotype 2 (8.87 [2.42] and 5.73 [1.03] points).The results suggest an association between temperament and enterotypes in healthy Korean adults. It is conceivable that this association may develop early in life as a result of biological processes catalyzed by the gut microbiota during infancy.