This study investigates the localization and immunoexpression levels of nesfatin-1 and ghrelin – two metabolically active peptides – and their putative shared receptor, growth hormone secretagogue receptor (GHSR), across the gastrointestinal tract (GIT) of Holstein-Friesian bulls. Recognized for their opposing roles in energy balance, nesfatin-1 and ghrelin are considered “sibling peptides” due to their complementary physiological functions and origin within the gastrointestinal system. The investigation encompassed both immature (calves) and mature (adult) cattle to assess developmental variation in the immunoexpression and localization of these peptides. Immunohistochemistry and ELISA were used to determine their localization patterns and quantify protein concentrations across distinct GIT segments. Nesfatin-1 was found broadly distributed in mucosal layers and the enteric nervous system (ENS), with a pronounced presence in the abomasum and duodenum. Notably, calves exhibited higher levels of nesfatin-1 across most GIT regions, suggesting age-related differences in metabolic regulation. Ghrelin was predominantly localized in the abomasum and, to a lesser extent, in other gastrointestinal regions, including the forestomachs and intestinal mucosa. Its presence in neuronal structures of the ENS, although less abundant, hints at potential neural roles beyond endocrine signalling. GHSR immunoexpression was restricted mainly to the enteric ganglia and selected epithelial cells, with significant levels observed in the duodenum, particularly in calves. The receptor was absent in the rumen, implying that ghrelin activity in this region might be mediated via systemic or paracrine pathways rather than local receptor binding. The findings reveal both overlapping and distinct localization patterns of these peptides and their receptor, showing complex interactions in GIT physiology. Elevated nesfatin-1 immunoexpression in young animals suggests a potential developmental role, while the conserved ghrelin distribution reinforces its established gastric functions. These results may contribute insights into the regulatory architecture of bovine metabolism and potentially inform strategies for optimizing cattle growth and health management, providing a relevant reference point for veterinary sciences.