Ghrelin is a gastric hormone that stimulates hunger and worsens glucose metabolism. Circulating ghrelin is decreased after Roux-en-Y gastric bypass (RYGB) surgery; however, the mechanism(s) underlying this change is unknown. We tested the hypothesis that jejunal nutrient exposure plays a significant role in ghrelin suppression after RYGB. Feeding tubes were placed in the stomach or jejunum in 13 obese subjects to simulate pre-RYGB or post-RYGB glucose exposure to the gastrointestinal (GI) tract, respectively, without the confounding effects of caloric restriction, weight loss, and surgical stress. On separate study days, the plasma glucose curves obtained with either gastric or jejunal administration of glucose were replicated with intravenous (IV) infusions of glucose. These "isoglycemic clamps" enabled us to determine the contribution of the GI tract and post-absorptive plasma glucose to acyl ghrelin suppression. Plasma acyl ghrelin levels were suppressed to a greater degree with jejunal glucose administration compared to gastric glucose administration (P<0.05). Jejunal administration of glucose also resulted in a greater suppression of acyl ghrelin than the corresponding isoglycemic glucose infusion (P≤0.01). However, gastric and isoglycemic IV glucose infusions resulted in similar degrees of acyl ghrelin suppression (P>0.05). Direct exposure of the proximal jejunum to glucose increases acyl ghrelin suppression independent of circulating glucose levels. The enhanced suppression of acyl ghrelin after RYGB may be due to a nutrient-initiated signal in the jejunum that regulates ghrelin secretion.