Imatinib (Gleevec, STI571) is a drug used to treat certain types of cancer. Glucose-6-phosphate dehydrogenase and glutathione reductase are enzymes important for redox homeostasis and play key roles in many cellular processes. The purpose of the present work is to evaluate the in vitro effects of imatinib on sheep brain cortex glucose-6-phosphate dehydrogenase, and on bovine kidney cortex, bovine liver and yeast glutathione reductase. Kinetic studies on the inhibition of enzymes by imatinib have been investigated by using Lineweaver-Burk double reciprocal plot and values summarized with graphs by plotting the data using Linewear-Burk diagrams of 1/v against 1/[S] at each [I]. Imatinib inhibits glucose-6-phosphate dehydrogenase with an IC50 value of 0.7 mM. It inhibits bovine kidney cortex, liver and yeast glutathione reductase in a concentration-dependent manner with IC50 values of 0.8, 0.92, 1 mM, respectively. We have investigated the kinetic characteristics, inhibition types and constants (Ki). Inhibition of the glucose-6-phosphate dehydrogenase and glutathione reductase represents an attractive approach to the development of anticancer agents. This study shows the molecular effectiveness of the drug on purified enzymes of various sources. Understanding the kinetic mechanism of the drug and enzyme relationship may be a powerful approach to the future drug studies concerning new cancer drugs, drug resistance and new aspects in cancer therapy.