The hormone resistin is elevated in obesity and impairs glucose homeostasis. Here, we examined the effect of oligomerized human resistin on insulin signaling and glucose metabolism in skeletal muscle and myotubes. This was investigated by incubating mouse extensor digitorum longus (EDL) and soleus muscles and L6 myotubes with physiological concentrations of resistin and assessing insulin-stimulated glucose uptake, cellular signaling, suppressor of cytokine signaling 3 (SOCS-3) mRNA, and GLUT4 translocation. We found that resistin at a concentration of 30 ng/ml decreased insulin-stimulated glucose uptake by 30–40% in soleus muscle and myotubes, whereas in EDL muscle insulin-stimulated glucose uptake was impaired at a resistin concentration of 100 ng/ml. Impaired insulin-stimulated glucose uptake was not associated with reduced Akt phosphorylation or IRS-1 protein or increased SOCS-3 mRNA expression. To further investigate the site(s) at which resistin impairs glucose uptake we treated myotubes and skeletal muscle with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and found that, although resistin did not impair AMPK activation, it reduced AICAR-stimulated glucose uptake. These data suggested that resistin impairs glucose uptake at a point common to insulin and AMPK signaling pathways, and we thus measured AS160/TBC1D4 Thr642 phosphorylation and GLUT4 translocation in myotubes. Resistin did not impair TBC1D4 phosphorylation but did reduce both insulin and AICAR-stimulated GLUT4 plasma membrane translocation. We conclude that resistin impairs insulin-stimulated glucose uptake by mechanisms involving reduced plasma membrane GLUT4 translocation but independent of the proximal insulin-signaling cascade, AMPK, and SOCS-3.