Inflammation and airway hyperresponsiveness (AHR) are hallmark features of asthma and often correlate with the severity of clinical disease. Although these features of asthma can be effectively managed with glucocorticoid therapy, a subgroup of patients, typically with severe asthma, remains refractory to therapy. The mechanisms leading to steroid resistance in severe asthmatics are poorly understood but may be related to the activation of innate host defense pathways. Previously, we have shown that IFN-γ-producing cells and LPS, two factors that are associated with severe asthma, induce steroid-resistant AHR in a mouse model. We now demonstrate that cooperative signaling induced by IFN-γ and LPS results in the production of IL-27 by mouse pulmonary macrophages. IL-27 and IFN-γ uniquely cooperate to induce glucocorticoid-resistant AHR through a previously unknown MyD88-dependent mechanism in pulmonary macrophages. Importantly, integrated signaling by IL-27/IFN-γ inhibits glucocorticoid-induced translocation of the glucocorticoid receptor to the nucleus of macrophages. Furthermore, expression of both IL-27 and IFN-γ was increased in the induced sputum of steroid-refractory asthmatics. These results suggest that a potential mechanism for steroid resistance in asthma is the activation of MyD88-dependent pathways in macrophages that are triggered by IL-27 and IFN-γ, and that manipulation of these pathways may be a therapeutic target.