Background: Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) treatment enhance intestinal adaptation. To determine whether these growth factors exert synergistic effects on intestinal growth and function, GLP-2 and EGF-containing media (EGF-cm) were administered, alone and in combination, in neonatal piglet with short bowel syndrome (SBS). Methods: Neonatal Landrace/Large White piglets were block-randomized to 75% mid-intestinal (JI group) or distal-intestinal (JC group) resection or sham control, with 7-d infusion of saline (control), intravenous human GLP-2 (11 nmol/kg/day) alone, enteral EGF-cm (80 μg/kg/day) alone, or GLP-2 and EGF-cm in combination. Adaptation was assessed by intestinal length, histopathology, Üssing chamber analysis and RT-qPCR of intestinal growth factors. Results: Combined EGF-cm and GLP-2 treatment increased intestinal length in all three surgical models (p<0.01). EGF-cm alone selectively increased bowel weight per length and jejunal villus height in the JI group. The JC group demonstrated increased intestinal weight and villus height (p<0.01) when given either GLP-2 alone or in combination with EGF-cm, with no effect of EGF-cm alone. Jejunal permeability of mannitol and polyethylene glycol decreased with combination therapy in both SBS groups (p<0.05). No difference was observed in fat absorption or body weight gain. IGF-1 mRNA was differentially expressed in JI versus JC piglets with treatment. Conclusions: Combined treatment with GLP-2 and EGF-cm induced intestinal lengthening and decreased permeability, in addition to the trophic effects of GLP-2 alone. Our findings demonstrate the benefits of novel combination GLP-2 and EGF treatment for neonatal SBS, especially in the JC model representing most human infants with SBS.