Glutathione reductase (GR, EC 18.104.22.168) is an antioxidant enzyme and is involved in the reducing reaction of oxidized glutathione. Glutathione and reduced glutathione are very crucial for micro- and macroorganisms because of their barrier function against radicals. Therefore, the aim of this article was to purify GR and evaluate the relationship between purified GR and vitamins. GR was purified from bovine liver using 2',5'-ADP Sepharose 4B column materials. To check the purity of each subunit of the enzyme, SDS-PAGE electrophoresis was performed and each one was 55 kDa. Afterwards, specific activity and purified ratio for the enzyme were calculated as 54 EU/mg x protein and 2700 times, respectively. Vitamins have a regulatory role in organisms and also some, like vitamin B, are coenzymes due to having cofactor effects. In this project, variable concentrations of some water-soluble vitamins, riboflavin, ascorbic acid, nicotinamide, folic acid, and thiamine, were tested. As a result of these kinetic findings, nicotinamide and folic acid increased the activity of glutathione reductase, while thiamine decreased it. Riboflavin and ascorbic acid did not show a stable effect on enzyme activity. Ki and IC50 constants were found as 50.16 +/- 5.63 mM and 21.04 mM, respectively, when Lineweaver-Burk and activity % vs. inhibitor concentration graphs were drawn for thiamine. The results of this article illustrate that the behaviors of vitamins could be complicated and vary from one living organism to other; in other words, they are unpredictable.