Glucagon-like peptide-2 (GLP-2) is a nutrient-dependent, intestinotrophic hormone derived from posttranslational processing of proglucagon in the distal bowel. GLP-2 is thought to act through indirect mediators, such as insulin-like growth factor I (IGF-I). We investigated if intestinal expression of GLP-2 and IGF-I system components are increased with the mucosal growth induced by enteral nutrient (EN) and/or a low dose of GLP-2 in parentally-fed rats. Rats were randomized to 4 treatment groups using a 2x2 design and maintained with parental nutrition (PN) for 7 days: PN alone, EN, GLP-2, EN+GLP-2, n=7-9. The 2 main treatment effects are: ±GLP-2 (100 µg/kg body wt/day) and ±EN (43% of energy needs, day4-6). Combination treatment with EN+GLP-2 induced synergistic intestinal growth in ileum resulting in greater mucosal cellularity, sucrase segmental activity and gain of body weight (ENxGLP-2, p<0.04). In addition, EN+GLP-2 induced a significant 28% increase in plasma concentration of bioactive GLP-2, a significant 102% increase in ileal proglucagon mRNA with no change in ileal dipeptidyl peptidase-IV (DPP-IV) specific activity, and significantly reduced plasma DPP-IV activity compared to GLP-2. This indicates that EN potentiates the intestinotrophic action of GLP-2. Proliferation of enterocytes due to GLP-2 infusion was associated with greater expression of ileal proglucagon, GLP-2 receptor, IGF-I, IGF binding protein-3 mRNAs, and greater IGF-I peptide concentration in ileum (p<0.032). Ileal IGF-I mRNA was positively correlated with expression of proglucagon, GLP-2R, and IGFBP-5 mRNAs (R²=0.43-0.56, p<0.0001). Our findings support the hypothesis that IGF-I is one of the downstream mediators of GLP-2 action in a physiological model of intestinal growth.