11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) converts active 11β-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11β-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11β-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11β-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11β-HSD2 was potently inhibited by all three compounds (IC values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC values compared to human), and mouse and zebrafish 11β-HSD2 were very weakly inhibited (IC values above 7 μM). Sequence alignment and application of newly generated homology models for human and mouse 11β-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11β-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11β-HSD2. The results provide an explanation for the observed species-specific 11β-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11β-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies.