Gut hormones are key regulators of homeostasis, modulating food intake, digestion, intestinal transit or insulin secretion. They are secreted by a specific population of intestinal epithelial cells, the enteroendocrine cells. Different subpopulations of EECs have been described along the gastro-intestinal tract, resulting in a specific pattern of hormones produced in the different regions of the intestines, allowing for these hormones to have both local and systemic effects. Ageing has been associated with increased prevalence of metabolic diseases and deregulation of several functions regulated by gut hormones such as digestion, intestinal transit or food intake. However, the importance of gut hormones in this phenotype is not known. Little literature has assessed to our knowledge a change in gut hormone circulating levels in elderly patients. To precisely describe a potential effect of ageing on gut hormone productions, we applied a peptidomics approach to assess most gut peptides along the intestinal tract of aged (18 months) and young (3 months) rats. This method allows the precise identification of the peptide, as well as the quantification to compare hormone processing and production between different conditions. We also collected samples for gene expression analysis and fasting plasma to measure circulating GLP-1 and GIP, the two incretin hormones. Ageing was associated with a change in the expression and production of some hormones, such as an increased CCK and decreased GIP production in the duodenum or a decreased SST production. The importance of these changes as well as the mechanisms involved in these changes remain to be deciphered.