Keratinocytes make a stratified epidermoid structure when cultured at an air-liquid interface. The three-dimensional (3D) culture of keratinocytes has been successfully used for more than 25 years, but it is still unclear why keratinocytes stratify in response to air exposure. AP-1 proteins are ubiquitous transcription factors that regulate many biological processes, including cell proliferation, differentiation and apoptosis. We established HaCaT-JunBDeltaN, a human keratinocyte cell line that expressed a mutant JunB with a dominant negative effect on AP-1 activity. Stratification of HaCaT-JunBDeltaN cells was markedly suppressed in a 3D culture condition, in which HaCaT cells stratified similarly to stratified squamous epithelia. However, HaCaT-JunBDeltaN cells had proliferation activities that were closely equivalent to those of HaCaT cells, under both two-dimensional (2D) and 3D culture conditions. To screen for the candidate gene responsible for the different stratification ability, we examined the gene expression profile of HaCaT cells before and after air exposure. Several genes with an antioxidative function, such as aldo-keto reductase and selenoprotein P were highly expressed after air exposure in HaCaT cells but not in HaCaT-JunBDeltaN cells. Our findings indicate the presence of a novel role of AP-1 activity when HaCaT cells make a stratified epidermoid structure under 3D culture conditions.