The present study was designed to identify changes in human serum caused by myosin light chain kinase (MLCK) in the context of Type 2 diabetes mellitus (T2DM), and to determine whether it had correlations with other biomarkers of T2DM. A total of 336 patients with T2DM and 90 sex- and age-matched, apparently healthy subjects were selected. The serum MLCK of all participants was detected by quantitative ELISA. Our results showed that the concentration of MLCK in the T2DM group was significantly higher than in control samples, and there were no correlations with age, FBG, TC, HDL-C, LDL-C or HbA1c. These findings suggest that serum MLCK is associated with T2DM, which gives some clues for identifying new biomarkers for the diagnosis of T2DM and its complications.