The initiation of enteral feeding represents a challenge to the neonatal intestinal microcirculation, especially in preterms where it predisposes to necrotizing enterocolitis (NEC). We hypothesized that a structural microvascular deficiency may occur when enteral feeding is initiated in preterm piglets susceptible to NEC. Stereologic volume densities of a pan-endothelial marker (vWF), and the main vasodilator endothelial nitric oxide synthase (eNOS), were determined along the small intestine of 1) unfed preterm piglets, 2) piglets receiving total parenteral nutrition (TPN) for 2-3 d, and 3) piglets fed 2 d sow's colostrum (TPN+SOW) or milk formula (TPN+FOR) following TPN. In the mucosa, vWF-density decreased in a cranio-caudal direction. A corresponding mucosal eNOS gradient appeared only after initiating enteral feeding. In TPN+SOW, eNOS induction may lag behind the mucosal growth of the caudal region. In TPN+FOR, formula-related factors (i.e. bacteria, cytokines) may suppress mucosal eNOS, indicated by increased stress-sensitive nuclear HIF1alpha staining. The low mucosal endothelial eNOS density was related to the presence of NEC lesions, maybe via increased hypoxia-sensitivity, especially in the caudal region as indicated by nuclear HIF1alpha-staining. Our results suggest an insufficient structural adaptation of the microvasculature to enteral feeding, especially of mucosal eNOS, which may lead to NEC. AD - Department of Veterinary Medicine [E.R.H, M.O., A.L.M.W., C.J.G.], University of Antwerp, 2610 Wilrijk, Belgium; Department of Human Nutrition [T.T., P.T.S.], University of Copenhagen, Frederiksberg DK-1958, Denmark; Department of Pharmacology, Toxicology, Biochemistry and Organ Physiology [S.U.S.], University of Ghent, 9820 Merelbeke, Belgium; Department of Anatomy and Embryology [W.H.L.], University of Maastricht, 6200 MD Maastricht, The Netherlands.