Non-starch polysaccharides (NSPs) in cereals, as the main components of dietary fiber, have attracted widespread attention due to their physiological functions in regulating starch digestion rate and improving postprandial glycemic response in vivo. This paper systematically reviews the classification and spatial distribution of NSPs within the cereal grain matrix, and emphatically elaborates on the physicochemical and gut microbiota-mediated mechanisms of NSPs in glycemic regulation. It elucidates the key influencing factors of NSPs on glycemic regulation from the perspectives of differences in structural properties such as solubility, molecular weight and degree of polymerization, glycosidic bond types, and degree of cross-linking, as well as their different existing forms in the food matrices. Based on this, the paper proposes strategies to enhance the functional application of NSPs from the perspective of cereal materials selection, processing technology optimization, and multi-component formulation design. This review provides a theoretical basis and practical guidance for the development of functional cereal foods and the dietary management of chronic metabolic diseases.