Yin-Yang-1 (YY1) is a member of the GLI-Krüppel family of transcription factors, and both YY1 mRNA and protein expression have been identified in a number of different tissues and cell types suggesting that it is expressed both constitutively and ubiquitously. In epidermal tissue, however, we reported previously that YY1 protein is expressed at high levels in undifferentiated basal keratinocytes and is downregulated during differentiation toward the suprabasal layers. This differential expression pattern during keratinocyte differentiation suggests that YY1 may have an important role in regulating keratinocyte differentiation. In this study, we examined the role of YY1 in differentiation of the human keratinocyte cell line HaCaT using air-liquid interface three-dimensional culture. The constitutive overexpression of YY1 in HaCaT cells during air exposure-induced differentiation resulted in an undifferentiated phenotype, thickening of the stratified layers, suppression of differentiation marker expression, and retention of proliferative activity. These findings suggested that YY1 may have an important role in maintenance of the undifferentiated phenotype of keratinocytes in the basal epidermal layer, and that reduction of YY1 expression in the suprabasal layers may allow keratinocytes to differentiate and move toward the upper layers of the epidermis.