β-Glucans, dietary polysaccharides coming from a wide variety of sources, have been researched intensively. Being resistant to oral and gastrointestinal digestion, β-glucans can reach the colon where they are fermented by the gut microbiota, generating health-benefiting metabolites and demonstrating excellent prebiotic properties. This article reviews the diverse sources and structures of β-glucans, elucidates their functional mechanisms in regulating gut and systemic health, and summarizes the structure-activity relationship of β-glucans as prebiotics. The review aims to provide a theoretical foundation for the application of β-glucans.