Human milk, the best food for infants, is a dynamic and complex fluid that directly influences the immune system and microbiota establishment. The protective role of human milk is well known although the mechanisms behind it still need to be uncovered. This study aimed to characterize the impact of human milk in the immature intestine of newborns by analyzing the global transcriptomic response of the FHs 74 int cell line (ATCC CCL-241). The expression of intestinal keratins and other genes with a well-annotated intestinal or epithelial function validated FHs 74 int derived from the fetal small intestine as a model of the intestinal epithelium of newborns. Cells exposed to skimmed human milk showed seventeen differentially expressed genes, most of them up-regulated, including four chemokine genes (CXCL1, CXCL2, CXCL3 and CXCL10) and other immune-related genes. qRT-PCR and ELISA analysis confirmed the microarray data and indicated a different pattern of expression upon milk exposure in FHs 74 int as compared to the adult tumorigenic Caco-2 cell line. The evaluation of the functional significance of these transcriptomic changes reveals that human milk exposure may contribute to the regulation of the inflammatory response in the intestine during the perinatal period, which is characterized by the immaturity of the immune system and a pro-inflammatory phenotype.