Abstract:Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)‐dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue–green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2′,5′‐adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04‐fold over the crude extract, with a 19% yield, and specific activity of 0.7640 U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (Km) and maximum velocity of enzyme (Vmax) values for NADPH and 5,5′‐dithiobis(2‐nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+, Cu2+, Mg2+, Ni2+, Pb2+, Zn2+, Al3+, Cr3+, Fe3+, and V4+ ions; vitamin B3, vitamin B6, vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole‐3‐butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1‐chloro‐2,4‐dinitrobenzene (a positive control).