The incidence of eosinophilic esophagitis (EoE) has increased in the past several years, yet our understanding of its pathogenesis remains limited. To test the hypothesis that microRNAs (miRNAs) are altered in children with EoE, miRNAs were profiled in esophageal mucosa biopsies obtained from patients with active disease (n = 5) and healthy control subjects (n = 6). Fourteen miRNAs were significantly altered between groups; four of these miRNAs were decreased in EoE patients. A panel of five miRNAs (miR-203, miR-375, miR-21, miR-223, and miR-142-3p) were selected for validation in an independent set of samples from control (n = 22), active disease (n = 22), inactive disease (n = 22), and gastroesophageal reflux disease (n = 6) patients. Each panel miRNA was significantly altered among groups. miRNA changes in esophageal biopsies were not reflected in the circulating RNA pool, as no differences in panel miRNA levels were observed in sera collected from the four patient groups. In addition, in contrast to previous studies, no change in esophageal miRNA levels was detected following treatment that resolved esophageal eosinophilia. In an effort to identify the ramifications of reduced esophageal miR-203, miR-203 activity was inhibited in cultured epithelial cells via expression of a tough decoy miRNA inhibitor. Luciferase reporter assays demonstrated that miR-203 does not directly regulate human IL-15 through targeting of the IL-15 3′-untranslated region. From these experiments, it is concluded that miRNAs are perturbed in the esophageal mucosa, but not the serum, of pediatric EoE patients. Further investigation is required to decipher pathologically relevant consequences of miRNA perturbation in this context.