Endogenous glucocorticoid (GC) activation is regulated by the intracellular GC-activating and -inactivating enzymes 11β-hydroxysteroid dehydrogenase (11β-HSD)1 and 11β-HSD2, respectively, that catalyze interconversion of inert cortisone and its bioactive metabolite cortisol. Because endogenous GCs are critically implicated in suppressing the asthmatic state, this study examined the roles of the 11β-HSD enzymes in regulating GC activation and bronchoprotection during proasthmatic stimulation. Airway hyperresponsiveness to methacholine and inflammation were assessed in rabbits following inhalation of the proasthmatic/proinflammatory cytokine IL-13 with and without pretreatment with the 11β-HSD inhibitor carbenoxolone (CBX). Additionally, IL-13-induced changes in 11β-HSD isozyme expression and GC metabolism were examined in epithelium-intact and -denuded tracheal segments and peripheral lung tissues. Finally, the effects of pretreatment with CBX or 11β-HSD2-targeted siRNAs were investigated with respect to cortisol prevention of IL-13-induced airway constrictor hyperresponsiveness and eotaxin-3 production by airway epithelial cells. IL-13-exposed rabbits exhibited airway hyperresponsiveness, inflammation, and elevated bronchoalveolar lung fluid levels of eotaxin-3. These responses were inhibited by pretreatment with CBX, suggesting a permissive proasthmatic role for 11β-HSD2. Supporting this concept, extended studies demonstrated that 1) IL-13-treated tracheal epithelium and peripheral lung tissues exhibit upregulated 11β-HSD2 activity, 2) the latter impairs cortisone-induced cortisol accumulation and the ability of administered cortisol to prevent both IL-13-induced heightened airway contractility and eotaxin-3 release from epithelial cells, and 3) these proasthmatic responses are prevented by cortisol administration in the presence of 11β-HSD2 inhibition. Collectively, these data demonstrate that the proasthmatic effects of IL-13 are enabled by impaired endogenous GC activation in the lung that is attributed to upregulation of 11β-HSD2 in the pulmonary epithelium.