Diarrheal disease is a major cause of morbidity and mortality in infants and children worldwide. Evidence suggests that the interaction of immature human enterocytes with bacteria and their enterotoxins may account for the increased susceptibility of neonates to diarrheal diseases. However, the precise mechanisms that contribute to the excessive response to cholera toxin by the immature gut are largely unknown. Our aim was to characterize the cellular/molecular changes in Gs during gut development. In this study a colonic human epithelial cell line (T84) was used as representative of a mature enterocyte and a human fetal primary small intestinal cell line (H4) as representative of an immature enterocyte. Using our cell culture model of human intestinal development, we provide consistent evidence that CT-mediated Gs activation in fetal enterocytes differs from that of mature enterocytes and the difference may be related to ADP-ribosylation factor (ARF) interaction with the CT-signaling process. Here we demonstrated that ARF1 may play a critical role in clathrin-mediated CT-trafficking through the ER and Golgi and ARF6 may facilitate clathrin-mediated CT endocytosis that leads to enhanced Gs activation by CT. Collectively, these findings support our hypothesis that there is a developmentally regulated intestinal cellular response to bacterial exotoxins involving complex cellular events that accounts for the increased incidence and severity of toxigenic diarrhea during infancy.