Selenoproteins, i.e., proteins containing selenocysteine (Sec), are found in organisms of all three domains of life, Eukarya, Bacteria and Archaea. Sec is mostly present in the catalytic site of redox-active enzymes with diverse biological functions. The pathway for Sec synthesis and incorporation into proteins during translation differs from that of the 20 canonical amino acids, as Sec is synthesized on its transfer RNA (tRNA) and encoded by UGA, which normally signals termination of translation. The pathway of Sec biosynthesis and incorporation is well understood in Gram-negative bacteria, but considerable gaps of knowledge still exist in the respective systems of Gram-positive bacteria and Archaea. This chapter summarizes prokaryotic selenoprotein synthesis and function with a focus on emphasizing the differences between bacterial and archaeal pathways.