AbstractRelations between the gut microbiota and host mental health have been suggested by a growing number of case–control and cross-sectional studies, while supporting evidence is limited in large community samples followed during an extended period. Therefore, the current preregistered study (https://osf.io/8ymav, September 7, 2022) described child gut microbiota development in the first 14 years of life and explored its relations to internalizing and externalizing difficulties and social anxiety in puberty, a period of high relevance for the development of mental health problems. Fecal microbiota composition was analysed by 16S ribosomal RNA gene amplicon sequencing in a total of 1003 samples from 193 children. Through a clustering method, four distinct microbial clusters were newly identified in puberty. Most children within three of these clusters remained in the same clusters from the age of 12 to 14 years, suggesting stability in microbial development and transition during this period. These three clusters were compositionally similar to enterotypes (i.e., a robust classification of the gut microbiota based on its composition across different populations) enriched in Bacteroides, Prevotella, and Ruminococcus, respectively. Two Prevotella 9-predominated clusters, including one reported by us earlier in middle childhood and the other one in puberty, were associated with more externalizing behavior at age 14. One Faecalibacterium-depleted pubertal cluster was related to more social anxiety at age 14. This finding was confirmed by a negative cross-sectional relation between Faecalibacterium and social anxiety in the 14-year-olds. The findings of this study continue to map gut microbiota development in a relatively large community sample followed from birth onwards, importantly extending our knowledge to puberty. Results indicate that Prevotella 9 and Faecalibacterium may be relevant microbial taxa in relation to externalizing behavior and social anxiety, respectively. These correlational findings need validations from other similar cohort studies, as well as well-designed mechanistic pre-clinical investigations before inferring cause and effect.