AbstractZonula occludens‐1 (ZO‐1) is a scaffolding protein of tight junctions, which seal adjacent epithelial cells, that is also expressed in adherens junctions. The distribution pattern of ZO‐1 differs among stratified squamous epithelia, including that between skin and oral buccal mucosa. However, the causes for this difference, and the mechanisms underlying ZO‐1 spatial regulation, have yet to be elucidated. In this study, we showed that epithelial turnover and proliferation are associated with ZO‐1 distribution in squamous epithelia. We tried to verify the regulation of ZO‐1 by comparing normal skin and psoriasis, known as inflammatory skin disease with rapid turnover. We as well compared buccal mucosa and oral lichen planus, known as an inflammatory oral disease with a longer turnover interval. The imiquimod (IMQ) mouse model, often used as a psoriasis model, can promote cell proliferation. On the contrary, we peritoneally injected mice mitomycin C, which reduces cell proliferation. We examined whether IMQ and mitomycin C cause changes in the distribution and appearance of ZO‐1. Human samples and mouse pharmacological models revealed that slower epithelial turnover/proliferation led to the confinement of ZO‐1 to the uppermost part of squamous epithelia. In contrast, ZO‐1 was widely distributed under conditions of faster cell turnover/proliferation. Cell culture experiments and mathematical modelling corroborated these ZO‐1 distribution patterns. These findings demonstrate that ZO‐1 distribution is affected by epithelial cell dynamics.