We examined the relationships of glucose and HbA1c levels with the routinely screened serum enzyme activities in type 2 diabetes mellitus, and we designed an in vitro study to evaluate the direct effect of glucose levels on enzyme activities. The study was performed on a consecutive series of outpatients with type 2 diabetes who were followed up at Dicle University Medical Faculty Hospital from May 2009 to May 2010 for the first time. Effects of aspartate transaminase, aminotransferase, gamma‐glutamyl transferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities, glucose and HbA1c levels and in vitro glucose (492, 287, 184, 131, 82 mg dl^−1^, respectively) on enzymes were determined. The patients were categorized on the basis of glucose and HbA1c levels and grouped according to a range of values. In patients with high HbA1c levels (>10.1%), ALP, GGT activities and creatine kinase (CK)‐MB/CK (p = 0.008, 0.026, 0.014) ratio were increased significantly when compared with those in the control group. In patients with high glucose levels (>200 mg dl^−1^), ALP, GGT activities and CK‐MB/CK ratio (p = 0.003, 0.001, 0.001) were increased significantly when compared with those in the control group. Glucose, which was added to serum in different concentrations in vitro, did not directly affect enzyme activities such as ALP, GGT and CK. We concluded that increased glucose levels could damage the liver and the heart muscle cells. Monitoring of blood glucose levels is a more valuable parameter than monitoring HbA1c in the momentary evaluation of diabetes. Copyright © 2011 John Wiley & Sons, Ltd.