HIV-infected patients with lipodystrophy (HIV lipodystrophy) are insulin resistant and have elevated plasma free fatty acid (FFA) concentrations. We aimed to explore the mechanisms underlying FFA-induced insulin resistance in patients with HIV lipodystrophy. Using a randomized, placebo-controlled, cross-over design, we studied the effects of an overnight acipimox-induced suppression of FFAs on glucose and FFA metabolism by using stable isotope-labeled tracer techniques during basal conditions and a two-stage euglycemic-hyperinsulinemic clamp (20 and 50 mU insulin/m2 per min, respectively) in nine patients with nondiabetic HIV lipodystrophy. All patients received antiretroviral therapy. Biopsies from the vastus lateralis muscle were obtained during each stage of the clamp. Acipimox treatment reduced basal FFA rate of appearance by 68.9% (95% CI 52.6-79.5) and decreased plasma FFA concentration by 51.6% (42.0-58.9) (both, P < 0.0001). Endogenous glucose production was not influenced by acipimox. During the clamp, the increase in glucose uptake was significantly greater after acipimox treatment compared with placebo (acipimox: 26.85 μmol · kg-1 · min -1 [18.09-39.86] vs. placebo: 20.30 μmol · kg-1 · min-1 [13.67-30.13]; P < 0.01). Insulin increased phosphorylation of Akt Thr308 and glycogen synthase kinase-3β Ser9, decreased phosphorylation of glycogen synthase (GS) site 3a + b, and increased GS activity (percent I-form) in skeletal muscle (P < 0.01). Acipimox decreased phosphorylation of GS (site 3a + b) (P < 0.02) and increased GS activity (P < 0.01) in muscle. The present study provides direct evidence that suppression of lipolysis in patients with HIV lipodystrophy improves insulin-stimulated peripheral glucose uptake. The increased glucose uptake may in part be explained by increased dephosphorylation of GS (site 3a + b), resulting in increased GS activity.