Thioredoxin reductase (TrxR, EC 1.6.4.5) is a homodimeric flavoenzyme that catalyzed the reduction of the thioredoxin (Trx). It has been characterized in many bacteria and mammalian organism and categorized under two different types: bacterial and mammalian. Mammalian TrxRs contain an essential selenocysteine residue in the conserved C-terminal sequence. Mammalian TrxRs have three isoforms; cytosolic, mitochondrial and testis-specific. TrxR3, as testis-specific form, have different property than the other mammalian TrxRs. TrxR is involved in many cellular functions including DNA synthesis, redox signaling, antioxidative defence, selenium metabolism and regulation of apoptosis. Because of the many known functions, it is not surprising that this enzyme is a major subject of the many research. TrxR has been purified and characterized from a wide variety of species by using ion-exchange and affinity chromatographies. Notably, TrxR is a target enzyme for cancer drug research due to the relation with apoptosis. In this review we will present the intra- and extracellular biochemical functions of the enzyme and important medical applications in drug development.