We previously demonstrated the dose-dependent GLP-2 activation of intracellular signals associated with increased epithelial cell survival and proliferation in the neonatal intestine. Our current aim was to quantify the acute, temporal GLP-2 activation of these key intracellular signals and relate this to changes in epithelial cell survival and proliferation in the neonatal intestine. We studied twenty-nine TPN-fed neonatal piglets infused intravenously with either saline (control) or human GLP-2 (415 µmol·kg^-1·h^-1) for 1, 4 or 48 h. GLP-2 infusion increased small intestinal weight, DNA and protein content, and villus height at 48 h, but not at 1 or 4 h. Intestinal crypt and villus apoptosis decreased and crypt cell proliferation and protein synthesis increased linearly with duration of GLP-2 infusion, but were statistically different from controls only after 48 h. Prior to the morphological and cellular kinetic changes, GLP-2 rapidly activated putative GLP-2R downstream signals within 1-4 h, including phosphorylation of PKA, PKB, ERK1/2 and the transcription factors CREB and c-Fos. GLP-2 rapidly suppressed caspase-3 activation and upregulated Bcl-2 abundance within 1 h, whereas there was an increase in apoptosis inhibitors, XIAP at 1 h and cIAP-2 at 4 and 48 h. We also show that the increased c-Fos and reduced active caspase-3 immunostaining after GLP-2 infusion was localized in epithelial cells. We conclude that GLP-2-induced activation of intracellular signals involved in both cell survival and proliferation occurs rapidly and precedes the trophic cellular kinetic effects that occur later in intestinal epithelial cells.