Transglutaminase (TGase) is a calcium-dependent enzyme which catalyzes the iso-peptide cross-link between peptide-bound glutamine and lysinein vivo.Though the cross-link is developed as a barrier function in the skin system, overexpression of this could invoke skin hyperkeratosis in psoriasis and roughness in aged skin. In former research, many strong irreversible TGase inhibitors failed application because of high cytotoxicity. We selected one peptide after primary screening of six synthetic peptides designed from domains of known TGase substrates. Then we attempted to reduce the size and finally obtained two tetrameric peptides. When we treated keratinocyte with these TGase inhibitors under calcium-induced differentiation, the formation of a cornified cell envelope (CE) was decreased to the same level of CE under proliferating conditions without cytotoxic effect. Therefore, we propose that these TGase inhibitors may be useful for solving the physiological hypercross-linking problems for pharmaceutical or cosmetic purposes.