The epidermal-derived "alarmins" high-mobility group box 1 (HMGB1) protein and interleukin-33 (IL-33) are upregulated in patients with atopic dermatitis. However, their capacity as pro-inflammatory cytokines and their derived effects on skin barrier regulation are poorly elucidated. We investigated the impact of HMGB1 and IL-33 on gene transcription, protein expression and epidermal differentiation across 3 distinct keratinocyte in vitro models. Primary keratinocytes from healthy donors were used in submerged monolayer cultures, 3D human epidermis equivalents and 3D human skin equivalents. All keratinocyte models underwent 96-h stimulation with HMGB1 (100 mu M) or IL-33 (100 ng/ml) using IL-4 (50 ng/ml) as positive control of regulation and vehicle as negative control. We found that HMGB1 and IL-33 downregulated transcription of several genes from members of the epidermal differentiation complex, including filaggrin. Furthermore, HMGB1 downregulated the expression of the encoded proteins in the upper epidermis. Finally, IL-33 and HMGB1 ultimately led to impaired epidermal growth and maturation. In conclusion, HMGB1 and IL-33 could play a significant role in the atopic dermatitis pathophysiology due to negative regulation of structural proteins, stratum corneum formation and epidermal growth.