We tested the hypothesis that the loss of hepatic nerves decreases peripheral insulin sensitivity. Surgical hepatic denervation (DN) was performed in 22 dogs ∼16 days before study; 7 dogs (Sham-Sal) had a sham procedure. A euglycemic hyperinsulinemic (1 mU · kg⁻¹ · min⁻¹; arterial insulin 35 ± 1 μU/ml in all dogs) clamp was performed in conscious dogs. From 0 to 90 min of the clamp, all dogs received the same treatment; then the DN dogs were divided into three groups. From 90 to 180 min, DN-PeA (n = 7) and DN-PoA (n = 7) groups received acetylcholine 2.5 μg · kg⁻¹ · min⁻¹ via peripheral or portal vein, respectively, and DN-Sal (n= 8) received no acetylcholine. During 150–180 min, the Sham-Sal, DN-Sal, DN-PeA, and DN-PoA groups exhibited glucose infusion rates of 12.4 ± 0.8, 9.3 ± 0.8 (P < 0.05 vs. Sham-Sal), 9.1 ± 0.1 (P < 0.05 vs. Sham-Sal), and 12.7 ± 1.6 mg · kg⁻¹ · min⁻¹; nonhepatic glucose uptakes of 11.5 ± 0.9, 8.9 ± 0.7 (P < 0.05 vs. Sham-Sal), 8.6 ± 0.9 (P < 0.05 vs. Sham-Sal), and 11.9 ± 1.7 mg · kg⁻¹ · min⁻¹; net hindlimb glucose uptakes of 18.4 ± 2.1, 13.7 ± 1.1 (P< 0.05 vs. Sham-Sal), 17.5 ± 1.9, and 16.7 ± 3.2 mg/min; and glucose utilization rates of 14.4 ± 1.4, 10.4 ± 0.8 (P < 0.05 vs. Sham-Sal), 9.8 ± 0.9 (P< 0.05 vs. Sham-Sal), and 13.6 ± 1.8 mg · kg⁻¹ · min⁻¹, respectively. DN caused peripheral insulin resistance, and intraportal but not peripheral acetylcholine restored insulin sensitivity.