Thioredoxin reductase (E.C 1.6.4.5.; TrxR) is an enzyme belonging to the flavoprotein family of pyridine nucleotide-disulfide oxidoreductases. In this study, mitochondrial TrxR enzyme was purified from rainbow trout mitochondria. Thanks to the 2 consecutive procedures (preparation of homogenate and 2',5'-ADP Sepharose 4B affinity chromatography), the enzyme, having the specific activity of 11.9 EU mg protein-1, was purified with a yield of 2.38% and 672-fold. The purity of the enzyme was monitored and the molecular weight of its subunits was calculated as 70 kDa by SDS-PAGE. The native molecular mass of the enzyme was found to be approximately 151 kDa by gel filtration chromatography. Characteristic and kinetic properties of the enzyme were also determined. Furthermore, Se4+, Cu2+, Co2+, Ni2+, Fe3+, and Al3+ metal ions' in vitro effects on mitochondrial TrxR purified from rainbow trout was investigated. While Se4+ ion increased the enzyme activity, all of the other metal ions used in this study showed an inhibitory effect.