Weaning affects intestinal development in mammals, and pigs are often used as an animal model for analyzing the metabolism and physiology of differentiating middle villus intestinal epithelial cells (DIECs). To assess the impact of weaning on the proteomes of DIECs, we weaned piglets on day 14 and collected their jejunum on days 0, 1, 3, 5, and 7 after weaning. Levels of proteins associated with (a) Golgi vesicle transport and protein glycosylation; (b) monosaccharide, lipid, phospholipid, and nucleotide metabolism; and (c) Krebs cycle and respiratory electron transport chain were decreased in DIECs after weaning. These results indicate that weaning decreases nutrient metabolism in DIECs. Moreover, these results suggest that dietary interventions (e.g.,supplementation with functional amino acids) are required to counter these changes.