Background: Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. Objective: We sought to investigate in vivo the functional role of ORMDL3 in disease inception. Methods: An Ormdl3-deficientmousewas generated andthe role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adenoassociated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. Results: Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6-mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum-associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen-induced allergic airways disease. Conclusions: This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.