BACKGROUND: Mast cells (MCs) are pleiotropic cells that accumulate in the esophagus of patients with eosinophilic esophagitis (EoE) and are thought to contribute to disease pathogenesis, yet their properties and functions in this organ are largely unknown. OBJECTIVE: We aimed to perform a comprehensive molecular and spatial characterization of esophageal MCs in EoE. METHODS: Esophageal biopsies obtained from patients with active EoE, patients with EoE in histologic remission, and individuals with histologically normal esophageal biopsies and no history of esophageal disease (i.e., control individuals) were subject to scRNA-sequencing, flow cytometry, and immunofluorescence analyses. RESULTS: We probed 39,562 single esophageal cells by single-cell RNA sequencing; approximately 5% of these cells were MCs. We identified dynamic MC expansion across disease states. During homeostasis, TPSAB1(high)AREG(high) resident MCs were mainly detected in the lamina propria and exhibited a quiescent phenotype. In patients with active EoE, resident MCs assumed an activated phenotype, and two additional pro-inflammatory MC populations emerged in the intraepithelial compartment, each linked to a proliferating MKI67(high) cluster. One pro-inflammatory activated MC population, marked as KIT(high)IL1RL1(high)FCER1A(low), was not detected in disease remission (termed transient MC), whereas the other population, marked as CMA1(high)CTSG(high), was detected in disease remission where it maintained an activated state (termed persistent MC). MCs were prominent producers of esophageal IL-13 mRNA and protein, a key therapeutic target in EoE. CONCLUSIONS: Esophageal MCs comprise heterogeneous populations with transcriptional signatures associated with distinct spatial compartmentalization and EoE disease status. In active EoE, they assume a pro-inflammatory state and locally proliferate, and remain activated and poised to re-initiate inflammation even during disease remission.