High fructose consumption has implicated in insulin resistance and metabolic syndrome. Fructose is a highly lipogenic sugar that has intense metabolic effects in liver. Recent evidences suggest that fructose exposure to other tissues has substantial and profound metabolic consequences predisposing toward chronic conditions such as type 2 diabetes. Since skeletal muscle is the major site for glucose utilization, in the present study we define the effects of fructose exposure on glucose utilization in skeletal muscle cells. Upon fructose exposure, the L6 skeletal muscle cells displayed diminished glucose uptake, glucose transporter type 4 (GLUT4) translocation, and impaired insulin signaling. The exposure to fructose elevated reactive oxygen species (ROS) production in L6 myotubes, accompanied by activation of the stress/inflammation markers c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2), and degradation of inhibitor of NF-κB (IκBα). We found that fructose caused impairment of glucose utilization and insulin signaling through ROS-mediated activation of JNK and ERK1/2 pathways, which was prevented in the presence of antioxidants. In conclusion, our data demonstrate that exposure to fructose induces cell-autonomous oxidative response through ROS production leading to impaired insulin signaling and attenuated glucose utilization in skeletal muscle cells.