Necrotizing enterocolitis (NEC) is a gastrointestinal complication of premature infants with high rates of morbidity and mortality. A comprehensive view of the cellular changes and aberrant interactions that underlie NEC is lacking. This study aimed at filling in this gap. We combine single-cell RNA sequencing (scRNAseq), T-cell receptor beta (TCRβ) analysis, bulk transcriptomics, and imaging to characterize cell identities, interactions, and zonal changes in NEC. We find an abundance of proinflammatory macrophages, fibroblasts, endothelial cells as well as T cells that exhibit increased TCRβ clonal expansion. Villus tip epithelial cells are reduced in NEC and the remaining epithelial cells up-regulate proinflammatory genes. We establish a detailed map of aberrant epithelial–mesenchymal–immune interactions that are associated with inflammation in NEC mucosa. Our analyses highlight the cellular dysregulations of NEC-associated intestinal tissue and identify potential targets for biomarker discovery and therapeutics.