Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3'- and 6'-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.