Selenium is a non-metal with some metalloid properties that is essential in human health mainly due to the key role of selenoproteins. Information on selenium bioavailability is important to derive dietary recommendations. The bioavailability of selenium in raw and prepared foods as well as its essentiality/toxicity depends on its chemical forms. Likewise, the 2000 report of the US Food and Nutrition Board suggested that most dietary selenium is highly bioavailable: >90% of selenomethionine is absorbed; selenocysteine appears to be absorbed very well; ≈100% of selenate is absorbed, but a significant fraction is lost in the urine; and >50% of selenite is absorbed (depending on luminal interactions) and is better retained than selenate. For this reason, analytical methodologies focused on the determination of different chemical forms of selenium in food are of key importance. Experiments with mice models suggested that selenium in food can shape gut microbiota diversity, richness and composition, increasing the presence of some health relevant taxa. Moreover, selenium supplementation can shape gut metabolome and plasma selenoproteins expression. In this chapter, we review the differential selenium bioavailability in raw and processed foods, the analytical methods available for determining chemical forms of selenium and the impact of selenium in food in the gut microbiota of mammals.