The aim of this study was to examine the effect of high dietary oxidized frying oil (OFO) on insulin and energy metabolism related enzymes in leukocytes of mice. Three groups of mice were fed basal diet containing fresh sunflower oil (Control), OFO fried for 12 and 24 hours. Plasma glucose level was significantly increased after OFO feeding, whereas plasma immuno-reactive insulin (IRI) and insulin staining in islets cells showed significant decrease. Furthermore cholesterol and triglycerides levels were decreased. The activities of malate dehydrogenase (MDH) were significantly reduced in cytosolic and mitochondrial fractions of leukocytes in OFO compared to the control group. Also cytosolic MDH/lactic dehydrogenase (LDH) activity ratio was significantly decreased as LDH showed no significant changes between groups. The results of this study demonstrated that OFO feeding significantly decreased IRI, insulin secretion from pancreas and the activity of MDH in leukocytes of mice, therefore hyperglycemia and glucose intolerance occurred.