Ciliary neurotrophic factor (CNTF) is a member of the gp130 receptor cytokine family recently identified as an antiobesity agent in rodents and humans by mechanisms that remain unclear. We investigated the impact of acute CNTF treatment on insulin action in the presence of lipid oversupply. To avoid confounding effects of long-term high-fat feeding or genetic manipulation on whole-body insulin sensitivity, we performed a 2-h Intralipid infusion (20% heparinized Intralipid) with or without recombinant CNTF pretreatment (Axokine 0.3 mg/kg), followed by a 2-h hyperinsulinemic-euglycemic clamp (12 mU/kg·min) in fasted, male Wistar rats. Acute Intralipid infusion increased plasma free fatty acid levels from 1.0 ± 0.1 to 2.5 ± 0.3 mm, which subsequently caused reductions in skeletal muscle (insulin-stimulated glucose disposal rate) and liver (hepatic glucose production) insulin sensitivity by 30 and 45%, respectively. CNTF pretreatment completely prevented the lipid-mediated reduction in insulin-stimulated glucose disposal rate and the blunted suppression of hepatic glucose production by insulin. Although lipid infusion increased triacylglycerol and ceramide accumulation and phosphorylation of mixed linage kinase 3 and c-Jun N-terminal kinase 1 in skeletal muscle, CNTF pretreatment prevented these lipid-induced effects. Alterations in hepatic and muscle insulin signal transduction as well as phosphorylation of c-Jun N-terminal kinase 1/2 paralleled alterations in insulin sensitivity. These data support the use of CNTF as a potential therapeutic means to combat lipid-induced insulin resistance.