Barrier dysfunction has been implicated in the pathophysiology of eosinophilic esophagitis (EoE). Transforming growth factor-β1 (TGF-β1), a potent pleiotropic molecule, is increased in EoE; however, no study has evaluated its influence on esophageal epithelial barrier. We hypothesized that TGF-β1 regulates barrier dysfunction in EoE. We aimed to determine the role of TGF-β1 in the epithelial barrier in models of EoE. To examine the impact of TGF-β1 on esophageal barrier, immortalized human esophageal epithelial (EPC2-hTERT) cells were exposed to TGF-β1 during the three-dimensional air-liquid interface (3D-ALI) model in vitro. TGF-β1 exposure diminished EPC2-hTERT barrier function as measured by transepithelial electrical resistance (TEER) and 3 kDa Fluorescein isothiocyanate dextran paracellular flux (FITC Flux), and hematoxylin and eosin (H&E) assessment revealed prominent cellular separation. In analysis of epithelial barrier molecules, TGF-β1 led to the specific reduction in expression of the tight-junction molecule, claudin-7 (CLDN7), and this was prevented by TGF-β-receptor I inhibitor. Short hairpin ribonucleic acid (shRNA)-mediated CLDN7 knockdown diminished epithelial barrier function, whereas CLDN7 overexpression resulted in protection from TGF-β1-mediated barrier dysfunction. In pediatric EoE biopsies CLDN7 expression was decreased and altered localization was observed with immunofluorescence analysis, and the TGF-β1 downstream transcription factor, phosphorylated SMAD2/3 (pSMAD2/3), was increased. Our data suggest that TGF-β1 participates in esophageal epithelial barrier dysfunction through CLDN7 dysregulation.Mucosal Immunology advance online publication 23 August 2017; doi:10.1038/mi.2017.72.