Background: Glucocorticoids (GC) deteriorates skin barrier function. In skin where 11β-hydroxysteroid dehydrogenase 2 which inactivates GC is deficient, GC can bind to mineralocorticoid receptor (MR) as well as GC receptor (GR). MR has been reported to be implicated in GC-induced delayed wound healing and epidermal atrophy. Objectives: To investigate whether GC binds to MR and contributes to skin barrier dysfunction and whether MR antagonism prevents GC-induced skin barrier dysfunction. Methods: Normal human epidermal keratinocytes (NHEKs) treated with GC were examined for the expression and intracellular localization of GR and MR. Treating NHEKs with GC and mifepristone (GR antagonist) or eplerenone (MR antagonist), keratinocyte differentiation markers were analyzed. In healthy adults (n=6), skin barrier function was measured after applicating GC with spironolactone cream (MR blocker) or vehicle. Results: In NHEKs, GC decreased the expression of GR but not MR. In contrast, GC induced the nuclear localization of both GR and MR, inhibited by their own antagonists, respectively. GC decreased the differentiation markers, recovered by eplerenone. In topical GC treated skin, co-treatment of MR blocker improved the stratum corneum integrity and barrier recovery compared to control albeit without significance. Conclusion: GC binds to MR as well as GR and contributes to skin barrier dysfunction. Therefore, MR blockers as well as GR blockers might prevent GC-induced skin barrier dysfunction.