Pre-clinical models indicate a causative nexus between mitochondrial oxidative stress and insulin resistance. However, the pathophysiological significance of this mechanism in humans remains unproven. Herein, we employed an invasive in vivo mechanistic approach in humans to manipulate mitochondrial redox state while assessing insulin action. To this end, we combined intravenous infusion of a lipid overload with intake of a mitochondria-targeted antioxidant (mtAO) in conjunction with insulin clamp studies. During lipid overload, insulin-stimulated muscle glucose uptake, as determined by the femoral arteriovenous balance technique, was increased by mtAO. At the muscle molecular level, mtAO did not affect canonical insulin signaling but enhanced insulin-stimulated GLUT4 translocation while decreasing the mitochondrial oxidative burden under lipid oversupply. Ex vivo studies revealed that mtAO ameliorated features of mitochondrial bioenergetics, including diminished mitochondrial H₂O₂ emission, in muscle fibres exposed to high intracellular lipid levels. These findings implicate mitochondrial oxidants in the development of lipid-induced insulin resistance in humans.