Necrotizing enterocolitis (NEC) is a deadly gastrointestinal disease of premature infants characterized by an exaggerated inflammatory response, dysbiosis of the gut microbiome, decreased epithelial cell proliferation, and gut barrier disruption. Here, we describe a novel in vitro model of human neonatal small intestinal epithelium (Neonatal-Intestine-on-a-Chip) that mimics key features of intestinal physiology by utilizing a combination of premature infant intestinal enteroids co-cultured with human intestinal microvascular endothelial cells within a microfluidic device. We used our Neonatal-Intestine-on-a-Chip to recapitulate NEC pathophysiology in an in vitro model system of the premature gut inoculated with infant-derived microbiota. This model, also known as NEC-on-a-Chip, emulates the prominent features of NEC, demonstrating significant upregulation of pro-inflammatory cytokines, decreased intestinal epithelial cell markers, reduced epithelial proliferation and disrupted epithelial barrier integrity. NEC-on-a-Chip provides a novel preclinical model of NEC, which may be used as a personalized medicine approach to test new therapeutics for this devastating disease.