Introduction The global swine industry faces significant challenges related to improving the survival and health of newborn piglets. Attention has come to β-hydroxy-β-methylbutyrate (HMB), a metabolite of leucine, for its potential in prenatal nutritional programming in sows, which can improve piglet body weight and support the development of the skeletal and digestive systems. The effects of prenatal HMB supplementation were investigated on the chemical coding of the enteric nervous system (ENS) in the small intestine of neonatal piglets. Material and Methods The experiment was conducted on piglets from 12 sows divided into a control and an experimental group. Sows in the experimental group received HMB at a dose of 0.2 g/kg body weight per day from day 70 to 90 of gestation. After parturition, one piglet from each litter was euthanised and parts of the duodenum, jejunum and ileum were exsected. Tissue sections were fixed in paraffin, reacted with anti–vasoactive intestinal peptide (VIP), anti–cocaine- and-amphetamine-regulated transcript (CART), anti–neuronal nitric oxide synthase (nNOS) and anti-substance P (SP) antibodies, and the immunoexpression of VIP, CART, nNOS and SP was determined histomorphometrically by calculating the area of fibres which were immunoreactive with each. Results Supplementation with HMB in sows caused significant changes in the ENS of newborn piglets, including an increase in the area of fibres reactive to CART and nNOS in certain layers and sections of the small intestine, and a decrease in the area of fibres reactive to SP and VIP. Conclusion The results indicate that prenatal supplementation with HMB in sows may significantly influence the functioning of the gastrointestinal tract in newborn piglets.