On the basis of results in cell cultures, rodents, and pigs, l-arabinose may inhibit intestinal sucrase activity and thereby delay sucrose digestion. The objective was to investigate the dose-response effects of l-arabinose on intestinal sucrase activity in vitro and glucose tolerance, appetite, and energy intake in humans. In vitro, Caco-2 cells were cultured for 21 d, homogenized, and used as an enzyme preparation with sucrose as substrate in concentrations from 7 to 280 mmol/L with 0.84, 1.4, and 2.8 mmol l-arabinose/L as inhibitor. Released glucose was measured after 30 min. In the human studies, 15 healthy men participated in a randomized, double-blind, crossover study. Sucrose beverages (75 g in 300 mL) supplemented with 0%, 1.3%, 2.7%, and 4% by weight of l-arabinose were tested at breakfast. Blood for the measurement of glucose, insulin, C-peptide, incretin hormones, and triacylglycerol was collected under fasting conditions and for 3 h postprandially. Postprandial appetite sensations and energy intake at lunch were registered. In vitro, the addition of l-arabinose resulted in uncompetitive inhibition of sucrase activity. In the human studies, supplementation with 4% l-arabinose produced an 11% lower glucose peak, a 33% lower and delayed insulin peak, a 23% reduction in the incremental area under the curve (iAUC) for insulin, a 23% lower and delayed C-peptide peak, a 9% reduction in the iAUC for C-peptide, a 53% increase in the iAUC for glucagon-like peptide-1 (GLP-1), and a 28% reduction in the iAUC for glucose-dependent insulinotropic polypeptide. No effects on triacylglycerol, gastrointestinal symptoms, appetite ratings, or energy intake were observed. l-Arabinose inhibits sucrase activity from Caco-2 cells; 4% l-arabinose in sucrose beverages reduces postprandial glucose, insulin, and C-peptide responses and enhances the GLP-1 response in humans without gastrointestinal adverse effects. This trial is registered at clinicaltrials.gov as NCT00302302.