Human milk oligosaccharides (HMO) have been shown to interact directly with immune cells. However, large quantities of HMO are required for intervention or clinical studies, but these are unavailable in most cases. In this respect, bovine milk is potentially an excellent source of commercially viable analogues of these unique molecules. In the present study, we compared the transcriptional response of colonic epithelial cells (HT-29) to the entire pool of HMO and bovine colostrum oligosaccharides (BCO) to determine whether the oligosaccharides from bovine milk had effects on gene expression that were similar to those of their human counterparts. Gene set enrichment analysis of the transcriptional data revealed that there were a number of similar biological processes that may be influenced by both treatments including a response to stimulus, signalling, locomotion, and multicellular, developmental and immune system processes. For a more detailed insight into the effects of milk oligosaccharides, the effect on the expression of immune system-associated glycogenes was chosen as a case study when performing validation studies. Glycogenes in the current context are genes that are directly or indirectly regulated in the presence of glycans and/or glycoconjugates. RT-PCR analysis revealed that HMO and BCO influenced the expression of cytokines (IL-1β, IL-8, colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF2), IL-17C and platelet factor 4 (PF4)), chemokines (chemokine (C-X-C motif) ligand 1 (CXCL1), chemokine (C-X-C motif) ligand 3 (CXCL3), chemokine (C-C motif) ligand 20 (CCL20), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 6 (CXCL6), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X3-C motif) ligand 1 (CX3CL1) and CXCL2) and cell surface receptors (interferon γ receptor 1 (IFNGR1), intercellular adhesion molecule-1 (ICAM-1), intercellular adhesion molecule-2 (ICAM-2) and IL-10 receptor α (IL10RA)). The present study suggests that milk oligosaccharides contribute to the development and maturation of the intestinal immune response and that bovine milk may be an attractive commercially viable source of oligosaccharides for such applications.