© 2015, American Society for Biochemistry and Molecular Biology Inc. All rights reserved. Inflammation and oxidative stress through the production of reactive oxygen species (ROS) are consistently associated with metabolic syndrome/type 2 diabetes. Although the role of Nox2, a major ROS-generating enzyme, is well described in host defense and inflammation, little is known about its potential role in insulin resistance in skeletal muscle. Insulin resistance induced by a high fat diet was mitigated in Nox2-null mice compared with wild-type mice after 3 or 9 months on the diet. High fat feeding increased Nox2 expression, superoxide production, and impaired insulin signaling in skeletal muscle tissue of wildtype mice but not in Nox2-null mice. Exposure of C2C12 cultured myotubes to either high glucose concentration, palmitate, or H2O2 decreases insulin-induced Akt phosphorylation and glucose uptake. Pretreatment with catalase abrogated these effects, indicating a key role for H2O2 in mediating insulin resistance. Down-regulation of Nox2 in C2C12 cells by shRNA prevented insulin resistance induced by high glucose or palmitate but not H2O2. These data indicate that increased production of ROS in insulin resistance induced by high glucose in skeletal muscle cells is a consequence of Nox2 activation. This is the first report to show that Nox2 is a key mediator of insulin resistance in skeletal muscle.