Reactive oxygen species (ROS) is an important regulator in cellular signaling transduction, and many previous studies have indicated that acute ROS stimulation improves insulin sensitivity in skeletal muscle. In the study, we found that chronic ROS treatment caused serious insulin resistance in C2C12 myotubes. Glucose uptake and consumption assay indicated that pretreatment with 80 mu M H2O2 for 2 h inhibited insulin-stimulated glucose uptake in C2C12 myotubes, and the reason for it, is that chronic H2O2 treatment decreased insulin-induced glucose transporter 4 (GLUT4) translocation from cell plasma to cell membrane. Moreover, Akt2 phosphorylation depended on insulin was reduced in C2C12 myotubes of chronic H2O2 treatment. Together, this study provides further demonstration that chronic ROS stress is associated with insulin resistance of skeletal muscle in the progression of type 2 diabetes. (C) 2016 Elsevier Inc. All rights reserved.