Human milk (HM) is associated with major short- and long-term health benefits for infants. However, infant formulas (IF), substitutes for HM, are widely used for infant nutrition. Although these IFs meet the nutritional needs of newborns, they are devoid of many bioactive compounds present in HM, such as immunoglobulins, hormones and a multitude of metabolites. HM metabolites include short chain fatty acids (SCFAs) (butyric acid, acetic acid, propionic acid), polyamines (putrescine, spermine, spermidine), tryptophan derivatives (indole, indole-lactic acid, kynurenine) as well as GABA and lactate. These metabolites are known to be produced within the gastrointestinal tract by the intestinal microbiota and to have effects on the host physiology but their relevance in HM has not been studied so far. The objective of our study was therefore to decipher the effects of HM metabolites on intestinal physiology using an in vitro pluricellular and polarized model of intestinal epithelium, including Caco-2 (as enterocytes), HT-29 MTX (as goblet cells), NCI-H716 (as enteroendocrine cells) and M cells. HM metabolites were studied at concentrations close to those found in HM, that is quite low. Their effects on trans-epithelial electric resistance (TEER) and on the expression of genes involved in the intestinal barrier, immune, antioxidant, endocrine and digestive functions were analyzed. SCFAs strongly modulated different intestinal functions, particularly the immune one with a significant downregulation of genes coding for IL-8, MyD88 and TFF3. They also modulated genes encoding tight junctions, as did GABA and polyamines, upregulating CLDN3, TJP1 and CLDN4 respectively and downregulating CLDN1 for SCFA and polyamines and CLDN7 for GABA. In parallel SCFAs significantly increased TEER, highlighting a potential reinforcing effect on the epithelial barrier while polyamines and GABA had no effect on TEER. Finally, SCFAs, GABA and lactic acid modulated the expression of some transporters involved in nutrition such as MCT1, GLUT1 and SGLT1 respectively. HM metabolites, despite their low concentration, are able to impact the intestinal barrier physiology, inviting us to consider their implementation in IF for a better mimicry of HM health benefits.