Prebiotics are nondigestible food components that have an impact on gut microbiota composition and activity, which in turn results in the improvement of health conditions. Nowadays, the production of prebiotics from agro-industrial by-products is under investigation. In this regard, polysaccharides are usually found in these sources and their potential use as prebiotics has been studied recently since these compounds act as substrates for the human gut microbiota, and they have the potential to modulate its composition through many mechanisms. Additionally, the use of agricultural by-products is advantageous because it is a cheap and abundantly available material. This review focuses on the recent scientific literature regarding the prebiotic properties of polysaccharides from agro-industrial by-products. PRACTICAL APPLICATIONS: Currently, the maintenance of gut homeostasis is a target for the improvement of human health. This review can broaden the perspective on the utilization of agro-industrial by-products that can compete in the market with the commercial ones or act as a source for new food ingredients.