Within the complex regulatory network of the gastrointestinal tract, enteroendocrine cells play a fundamental role in coordinating digestive processes and maintaining metabolic balance. These cells produce a wide range of hormones that influence secretion, motility, and nutrient utilization, constituting an essential component of gastrointestinal physiology in ruminants. Despite the growing interest in the role of endocrine regulation in digestive system function, comprehensive studies evaluating enteroendocrine cell populations across different segments of the gastrointestinal tract in cattle remain limited. In particular, the organization of these cell populations in key digestive segments such as the abomasum and small intestine requires further investigation to better understand their functional significance. The aim of the present study was to provide a comparative characterization of selected enteroendocrine cell populations in the abomasum and small intestine of young and adult Holstein–Friesian bulls. Tissue samples collected from the abomasum, duodenum, jejunum, and ileum were subjected to histological and immunohistochemical analyses. Chromogranin A (CgA) was used as a general marker of enteroendocrine cells, and the number of cells immunoreactive to CgA as well as to gastrin, secretin, somatostatin (SOM), glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1) was quantified in the examined segments of the gastrointestinal tract. All types of immunoreactive cells were counted using a standardized quantitative method based on the assessment of positively stained cells in randomly selected microscopic fields of histological sections. The results revealed clear differences in the number of enteroendocrine cells between the analyzed segments of the gastrointestinal tract as well as between the studied age groups. These findings indicate regional and age-related variation in enteroendocrine cell populations within the gastrointestinal tract and highlight their association with the functional organization of individual segments of the digestive system.