Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD as it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion- and basement membrane -proteins. Minimally expressed in normal skin, GzmB is elevated in AD and positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- to wild-type mice, showing significant reductions in inflammation, epidermal thickness and lesion formation in GzmB-/- mice. Topical administration of a small molecule GzmB inhibitor reduced disease severity compared to vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and filaggrin cleavage. Together, GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.