Arabinoxylans and mixed linkage (1 → 3)(1 → 4)-β- d-glucans are the major non-starch polysaccharides present in various tissues of barley. Depending on the genotypic or cellular origin, both polymers exhibit variations in their molecular structures. The molecular features of β-glucans and arabinoxylans are important determinants of their physical properties, such as water solubility, viscosity, and gelation properties as well as of their physiological functions in the gastro-intestinal tract. Barley β-glucans have been associated with lowering plasma cholesterol, reducing glycaemic index, and reducing risk of colon cancer. The potential application of β-glucans as food hydrocolloids has been also proposed based on their rheological characteristics. In addition to solution viscosity enhancement, β-glucans have been shown to gel under certain conditions. Arabinoxylans have been shown to significantly affect cereal-based processes such as milling, brewing, and breadmaking. Furthermore, arabinoxylans offer nutritional benefits of soluble and insoluble fibre, and, because of the presence of phenolic moieties in their molecular structures, they may also have some antioxidant properties.