Allergic asthma is an inflammatory disorder of the airway without satisfactory traditional therapies capable of controlling the underlying pathology. New approaches that can overcome the detrimental effects of immune dysregulation are thus desirable. Here we adoptively transfer ovalbumin (OVA) peptide-primed CD4-CD8- double negative T (DNT) cells intravenously into a mouse model of OVA-induced allergic asthma to find that OVA-induced airway hyperresponsiveness, lung inflammation, mucus production and OVA-specific IgG/IgE production are significantly suppressed. The immunosuppressive function of the OVA-specific DNT cells is dependent on the inhibition of CD11b+ dendritic cell function, T follicular helper cell proliferation, and IL-21 production. Mechanistically, Lag3 contributes to MHC-II antigen recognition and trogocytosis, thereby modulating the antigen-specific immune regulation by DNT cells. The effectiveness of ex vivo-generated allergen-specific DNT cells in alleviating airway inflammation thus supports the potential utilization of DNT cell-based therapy for the treatment of allergic asthma.