Mixed-linkage β-glucans (MLGs) are ubiquitous cell wall structural polysaccharides, mainly present in grasses and other organisms (i.e., Equisetaceae and lichens). MLGs are linear homopolysaccharides built by d-glucopyranose units (Glc) that show a singular primary structure of both β-(1→3) and β-(1→4) glycosidic linkages. This creates a block intramolecular copolymer structure with randomly distributed cellotriose, cellotetraose and even longer oligosaccharide domains, depending on the biological source. The solubility and aggregative properties of MLGs are largely dependent on their intramolecular structure, molecular weight, and concentration, which in turn influence their viscosity and rheological properties. MLGs are an important source of dietary fibers in our diets with nutritional and health benefits, contributing to improved cholesterol levels, blood sugar regulation, immunomodulatory function, satiety and weight management, and gut health as prebiotics. The mechanisms involved in these health benefits are related to both physicochemical and biological processes, such as increased viscosity of the digesta, interactions with starch and other food nutrients, inhibition of the enzyme activity, increased bile salt excretion, and enhanced growth of beneficial gut bacteria and production of bacterial metabolites. MLGs have important nutritional and biological applications as food ingredients in bread and baking applications, and in plant-based alternatives to animal products.