The gastrointestinal (GI) tract is a complex organ system crucial for digestion and nutrient absorption. The integrity of the gut barrier, particularly the mucus layer, is vital for maintaining health. This review explores the structure, composition, and regulation of the mucus layer, as well as its interactions with the gut microbiota and external factors such as diet and inflammation. **Mucus Structure and Organisation:** Mucins are large, glycosylated proteins that form the mucus layer, which is composed of water, electrolytes, lipids, and mucins. Mucins can be classified into transmembrane mucins and gel-forming mucins. Transmembrane mucins are anchored to the cell membrane, while gel-forming mucins are secreted by goblet cells and form a gel-like structure. **Mucin Synthesis and Degradation:** Mucin synthesis involves complex processes, including glycosylation, which varies along the GI tract and among individuals. Mucin degradation occurs through mechanical forces and microbial enzymes, affecting the mucus layer's protective function. **Role of Mucin:** The mucus layer protects the intestinal epithelium from mechanical, chemical, and biological stress. It also serves as a barrier for nutrient absorption and immune defense. In the small intestine, mucus is more permeable to bacteria, while in the large intestine, it forms a protective barrier against bacteria. **Bidirectional Interaction with Gut Microbiota:** The gut microbiota significantly influences mucus properties, including composition and thickness. Specific bacteria, such as *Akkermansia muciniphila*, contribute to mucus degradation and can affect mucus layer thickness. The composition of the gut microbiota varies from the mucosal to the luminal side, with different microbial communities in each region. **Mucus Layer Impairment:** Impairment of the mucus layer can lead to infections and inflammation. Pathogenic microorganisms can degrade mucus, allowing them to penetrate the intestinal epithelium. Diet, particularly high-fat and low-fiber diets, can disrupt the mucus layer, increasing permeability and susceptibility to infections. **Mucus Layer Enhancement:** Probiotics and beneficial bacteria can enhance the mucus layer by increasing mucin production and improving gut health. For example, *Lactobacillus* spp and *Bifidobacterium longum* have been shown to restore mucus layer thickness in experimental models. In conclusion, the mucus layer is a dynamic and complex structure that plays a crucial role in maintaining gut health. Understanding its regulation and interactions with the gut microbiota is essential for developing strategies to prevent and treat gastrointestinal diseases.The gastrointestinal (GI) tract is a complex organ system crucial for digestion and nutrient absorption. The integrity of the gut barrier, particularly the mucus layer, is vital for maintaining health. This review explores the structure, composition, and regulation of the mucus layer, as well as its interactions with the gut microbiota and external factors such as diet and inflammation. **Mucus Structure and Organisation:** Mucins are large, glycosylated proteins that form the mucus layer, which is composed of water, electrolytes, lipids, and mucins. Mucins can be classified into transmembrane mucins and gel-forming mucins. Transmembrane mucins are anchored to the cell membrane, while gel-forming mucins are secreted by goblet cells and form a gel-like structure. **Mucin Synthesis and Degradation:** Mucin synthesis involves complex processes, including glycosylation, which varies along the GI tract and among individuals. Mucin degradation occurs through mechanical forces and microbial enzymes, affecting the mucus layer's protective function. **Role of Mucin:** The mucus layer protects the intestinal epithelium from mechanical, chemical, and biological stress. It also serves as a barrier for nutrient absorption and immune defense. In the small intestine, mucus is more permeable to bacteria, while in the large intestine, it forms a protective barrier against bacteria. **Bidirectional Interaction with Gut Microbiota:** The gut microbiota significantly influences mucus properties, including composition and thickness. Specific bacteria, such as *Akkermansia muciniphila*, contribute to mucus degradation and can affect mucus layer thickness. The composition of the gut microbiota varies from the mucosal to the luminal side, with different microbial communities in each region. **Mucus Layer Impairment:** Impairment of the mucus layer can lead to infections and inflammation. Pathogenic microorganisms can degrade mucus, allowing them to penetrate the intestinal epithelium. Diet, particularly high-fat and low-fiber diets, can disrupt the mucus layer, increasing permeability and susceptibility to infections. **Mucus Layer Enhancement:** Probiotics and beneficial bacteria can enhance the mucus layer by increasing mucin production and improving gut health. For example, *Lactobacillus* spp and *Bifidobacterium longum* have been shown to restore mucus layer thickness in experimental models. In conclusion, the mucus layer is a dynamic and complex structure that plays a crucial role in maintaining gut health. Understanding its regulation and interactions with the gut microbiota is essential for developing strategies to prevent and treat gastrointestinal diseases.