Faecalibacterium duncaniae A2-165 is a rod-shaped, non-motile, and Extremely Sensitive to Oxygen microorganism, belonging to one of the most abundant genera in the human gut microbiome. A decreased abundance of Faecalibacterium species is correlated to Inflammatory Bowel Diseases (IBDs), highlighting this genus as a marker of gut health and a promising Next-Generation Probiotic. Many studies have demonstrated the anti-inflammatory effect of the most studied species, F. duncaniae A2-165, on mouse models of IBDs, associating the disease amelioration and suppression of inflammatory markers with large amounts of butyrate produced by the bacteria, and the presence of the MAM protein. However, many factors in F. duncaniae metabolism could contribute to its importance in the gut. To address this issue, this study characterized the proteome of F. duncaniae A2-165 in the stationary phase through a LC-MS/MS label-free proteomics approach.We quantified 1.280 proteins in total, corresponding to 44,7% of the in silico predicted proteome, with clear distinction between insoluble and soluble protein abunndaces. The subcellular localization prediction of the quantified proteins revealed 802 cytoplasmic proteins, 265 membrane proteins, six extracellular proteins, eight cell wall proteins, and 199 unknown localization proteins. Differential abundance analysis between insoluble and soluble fraction showed 290 proteins more abundant and 330 less abundant in the insoluble fraction. Functional analysis of these proteins showed a predominance of transporter proteins in the insoluble fraction, while metabolism of amino acids, carbohydrates and nucleotides were predominant in the soluble fraction. Further analysis of enriched pathways for each fraction showed energy metabolism, carbon cycle, and amino acid metabolism enriched in the soluble, and ABC transporters, quorum sensing, and oxidative phosphorylation in the insoluble. Moreover, we could confirm the presence of proteins associated to the bacteria’s mechanism of action, notably the key butyrate production pathway ButCoAT, the MAM protein and the ABC transporter exporter, and shikimate pathway enzymes.