BACKGROUND: The active form of vitamin D(3) , calcitriol, is widely used for the treatment of psoriasis, with or without topical corticosteroids. Topical corticosteroids are known to disrupt permeability and antimicrobial barriers, even with short-term use. Yet, the effect of topical calcitriol on epidermal permeability and antimicrobial barriers disrupted by topical corticosteroids has not been determined. OBJECTIVES: To examine the effect of calcitriol on epidermal permeability and antimicrobial barrier function that has been impaired by corticosteroids, as well as to elucidate the mechanism of improvement. MATERIAL AND METHODS: Topical calcitriol or the control vehicle was applied to each flank of hairless mice 20 min after treatment with topical clobetasol propionate and repeated every 12 h for 3·5 days. Barrier function assessment, Nile red staining, electron microscopy, immunohistochemistry, Western blotting, and real-time reverse transcriptase-polymerase chain reaction studies were performed 24 h after the last application. RESULTS: Epidermis co-treated with topical calcitriol showed an improvement of stratum corneum integrity and barrier recovery, more intense fluorescence staining with Nile red, and an increase in lamellar body (LB) maturation and density, as well as upregulation of major epidermal lipid synthesis-related enzymes (3-hydroxy-3-methylglutaryl-CoA, serine-palmitoyl transferase and fatty acid synthase), mouse beta-defensin 3, cathelin-related antimicrobial peptide and vitamin D receptor. CONCLUSIONS: We found that topical calcitriol restored both the epidermal permeability and antimicrobial barrier that had been impaired by corticosteroids. This restoration was mediated by both an activation of the cutaneous vitamin D pathway and an increase of epidermal lipids and antimicrobial peptides, promoted by the formation of the LB and the activity of epidermal lipid synthesis-related enzymes.