We tested whether the Th2 agonist and allergenic ligand IL-33, was associated with eosinophilic esophagitis (EoE) development in a paediatric cohort, and whether IL-33 protein could induce disease symptoms in mice. Biopsies from eosinophilic esophagitis patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast and epithelial cell markers, near blood vessels. Circulating levels of IL-33 were not increased. The time-course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model, and chronicity of IL-33 expression was demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 week, and esophageal pathology evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and up-regulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, were increased after IL-33 application, along with ILC2, Th1/17 and M2 macrophage marker genes. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with paediatric EoE development, and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.