Aims—A growing body of evidence suggests a role for altered epithelial barrier function in the pathophysiology of eosinophilic oesophagitis (EoE), but few have described the epithelial structure during inflammation. The purpose of this study was to define ultrastructural features of active, inactive EoE and control subject’s oesophageal epithelia. Methods—We prospectively enrolled patients undergoing diagnostic upper endoscopy for evaluation of EoE. Mucosal pinch biopsies were obtained from the distal oesophagus and processed for routine histology and electron microscopic assessment. Clinical features of enrolled subjects were analysed and subjects were divided into four groups: normal, gastroesophageal reflux disease (GERD), inactive EoE and active EoE. Representative photomicrographs of the basal and superficial epithelia were reviewed for abnormalities. Desmosomes were quantified on the surface of epithelia three to four prickle-cell layers above the basal layer. Results—Twenty-nine paediatric cases (ages 2–18 years) were enrolled in the study. We observed a significant decrease in the number of desmosomes per cell (DPC) of subjects with active EoE compared with inactive EoE, GERD and normal epithelia. With respect to DPC, no significant differences were found between inactive EoE compared with GERD or normal subjects. Additional ultrastructural features observed included epithelial microplicae and evidence of eosinophil transmigration, degranulation, and sombrero formation. Conclusions—Consistent with clinical and molecular findings, our ultrastructural data provide support for an altered oesophageal barrier in paediatric cases with active EoE, which may improve following treatment