Intestinal hormones stimulate more than 50% of the insulin response after oral glucose administration. Short chain fatty acids stimulate mucosal adaptation and may alter proglucagon messenger RNA and release of the insulin secretagogue, glucagon-like peptide-1 (GLP-1). Sprague-Dawley rats ingested a fiber-free elemetnal diet or an elemental diet supplemented with 30% fiber providing similar energy and nutrients for 14 days. The cecal and colonic short chain fatty acids contents were significantly higher in the 30% fiber group. Ileal proglucagon messenger RNA levels were significantly higher in the 30% group vs. the 0% group (11.47 +/- 0.87 vs. 6.52 +/- 0.87 densitometer units), respectively. Similar trends were seen in the colon (13.36 +/- 1.0 vs. 10.90 +/- 0.77 densitometer units; P = 0.07). Plasma GLP-1, insulin, and C peptide levels 30 min postoral glucose were significantly higher in the 30% fiber group vs. the 0% group (19.8 +/- 1.2 vs. 15.4 +/- 1.2 pg/ml, 2.67 +/- 0.4 vs. 1.29 +/- 0.5 ng/ml, and 964.4 +/- 94.4 vs. 530.2 +/- 120.4 pM, respectively). Plasma glucose and glucagon did not differ between groups. A diet supplemented with fiber is able to significantly alter proglucagon gene expression and modulate GLP-1 and insulin secretion. These novel findings deepen our understanding of the beneficial role of fiber in improving glucose homeostasis.