In the present study, the effect of methidathion, cypermethrin, and deltamethrin pesticides on Lake Van fish (Chalcalburnus tarichii Pallas, 1811) liver 6-phosphogluconate dehydrogenase enzyme activity was investigated due to the fact that these pesticides are extensively used to improve agricultural productivity in the Van region. 2′,5′-ADP Sepharose 4B affinity chromatography was used to purify 6-phosphogluconate dehydrogenase enzyme from fish liver and SDS-PAGE technique was used to control the purity of this enzyme. The in vitro effect of methidathion, cypermethrin, and deltamethrin pesticides on the enzyme activity was investigated. The enzyme was purified 1,050-fold with specific activity of 27.04 EU/mg protein. Moreover, Ki constants of methidathion, cypermethrin, and deltamethrin were to be 3.294 ± 0.215, 0.718 ± 0.095, and 0.084 ± 0.009 mM respectively. The IC 50 value were estimated as 9.95 × 10-5 ± 0.1844 × 10-5 mM for methidathion, 1.01 × 10-4 ± 0.01413 × 10-4 mM for cypermethrin, and 4.43 × 10-6 ± 0.05653 × 10-6 mM for deltamethrin. In conclusion, deltamethrin inhibits the enzyme activity more than methidathion and cypermethrin. © 2013 Springer Science+Business Media New York.