Insulin resistance correlates with intramyocellular lipid content (IMCL) and plasma free fatty acids (FFAs) and was recently linked to mitochondrial dysfunction. We examined the underlying relationships by measuring skeletal muscle ATP synthase flux, glucose transport/phosphorylation, and IMCL in response to different plasma insulin and plasma FFA concentrations. Healthy men were studied twice during hyperinsulinemic-euglycemic clamps with (LIP) or without (CON) lipid infusion (plasma FFA: CON ∼36 vs. LIP ∼1,034 μmol/l, P < 0.001). ATP synthase flux, glucose-6-phosphate (G6P), and IMCL were determined before and during the clamp in calf muscle using ³¹P and ¹H magnetic resonance spectroscopy. Plasma lipid elevation resulted in ∼46% reduced whole-body glucose metabolism (180–360 min; P < 0.0001 vs. CON) and a 70% lower rise of G6P (P < 0.05 vs. CON) without significant changes in IMCL (LIP 117 ± 12% vs. CON 93 ± 3% of basal, P = 0.073). During the clamp, ATP synthase flux increased by ∼60% under control conditions (P = 0.02 vs. baseline) and was 24% lower during lipid infusion (LIP 11.0 ± 0.9 vs. CON 14.6 ± 1.2 μmol · g muscle⁻¹ · min⁻¹, P < 0.05). Physiologically increased plasma FFA concentrations reduce insulin-stimulated muscle ATP synthase flux in parallel with induction of insulin resistance.