Abstract The aim of this study was to determine whether the nitric oxide (NO) synthase inhibitor, Ng-nitro-l-arginine-methyl-ester (l-NAME), reverses the effects of acute hyperglycaemia on gastric emptying and antropyloroduodenal (APD) motility. The study had a four-way randomized crossover (hyperglycaemia vs euglycaemia; l-NAME vs placebo) design in a clinical laboratory setting. Seven healthy volunteers [four males; age 30.3 ± 3.8 years; body mass index (BMI) 23.6 ± 1.2 kg m−2] were the study subjects. After positioning a transnasal manometry catheter across the pylorus, the blood glucose concentration was maintained at either 15 or 5 mmol L−1 using a glucose/insulin clamp. An intravenous infusion of l-NAME (180 μg kg−1 h−1) or placebo (0.9% saline) was commenced (T = −30 min) and continued for 150 min. At T = −2 min, subjects ingested a drink containing 50 g of glucose made up to 300 mL with water. Gastric emptying was measured using 3D ultrasound, and APD motility using manometry. Hyperglycaemia slowed gastric emptying (P < 0.05), and this effect was abolished by l-NAME. l-NAME had no effect on gastric emptying during euglycaemia. Hyperglycaemia suppressed fasting antral motility [motility index: 3.9 ± 0.8 (hyperglycaemia) vs 6.5 ± 0.6 (euglycaemia); P < 0.01]; l-NAME suppressed postprandial antral motility [motility index: 3.6 ± 0.2 (l-NAME) vs 5.1 ± 0.2 (placebo); P < 0.001]. Postprandial basal pyloric pressure was higher during hyperglycaemia (P < 0.001), and lower after administration of l-NAME (P < 0.001). Slowing of gastric emptying induced by hyperglycaemia is mediated by NO, and may involve the modulation of tonic pyloric activity.