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Gut microbiota is considered a separate organ with endocrine capabilities, actively contributing to tissue homeostasis. It consists of at least two separate microbial populations, the lumen-associated (LAM) and the mucosa-associated microbiota (MAM). In the present study, we compared LAM and MAM, by collecting stools and sigmoid brush samples of forty adults without large-bowel symptoms, and through a 16S rRNA gene next-generation sequencing (NGS) approach. MAM sample analysis revealed enrichment in aerotolerant <i>Proteobacteria</i>, probably selected by a gradient of oxygen that decreases from tissue to lumen, and in <i>Streptococcus</i> and <i>Clostridium</i> spp., highly fermenting bacteria. On the other hand, LAM microbiota showed an increased abundance in <i>Bacteroides, Prevotella</i>, and <i>Oscillospira</i>, genera able to digest and to degrade biopolymers in the large intestine. Predicted metagenomic analysis showed LAM to be enriched in genes encoding enzymes mostly involved in energy extraction from carbohydrates and lipids, whereas MAM in amino acid and vitamin metabolism. Moreover, LAM and MAM communities seemed to be influenced by different host factors, such as diet and sex. LAM is affected by body mass index (BMI) status. Indeed, BMI negatively correlates with <i>Faecalibacterium prausnitzii</i> and <i>Flavonifractor plautii</i> abundance, putative biomarkers of healthy status. In contrast, MAM microbial population showed a significant grouping according to sex. Female MAM was enriched in <i>Actinobacteria</i> (with an increased trend of the genus <i>Bifidobacterium</i>), and a significant depletion in <i>Veillonellaceae</i>. Interestingly, we found the species <i>Gemmiger formicilis</i> to be associated with male and <i>Bifidobacterium adolescentis</i>, with female MAM samples. In conclusion, our results suggest that gut harbors microbial niches that differ in both composition and host factor susceptibility, and their richness and diversity may be overlooked evaluating only fecal samples.