A multi-LU-domain-containing protein denoted C4.4A exhibits a tightly regulated membrane-associated expression in the suprabasal layers of stratified squamous epithelia such as skin and the esophagus, and the expression of C4.4A is dysregulated in various pathological conditions. However, the biological function of C4.4A remains unknown. To enable further studies, we evaluated the expression of C4.4A in monolayer cultures of normal human keratinocytes and in tissue-engineered skin substitutes (TESs) produced by the self-assembly approach, which allow the formation of a fully differentiated epidermis tissue. Results showed that, in monolayer, C4.4A was highly expressed in the centre of keratinocyte colonies at cell-cell contacts areas, while some cells located at the periphery presented little C4.4A expression. In TES, emergence of C4.4A expression coincided with the formation of the stratum spinosum. After the creation of a wound within the TES, C4.4A expression was observed in the suprabasal keratinocytes of the migrating epithelium, with the exception of the foremost leading keratinocytes, which were negative for C4.4A. Our results are consistent with previous data in mouse embryogenesis and wound healing. Based on these findings, we conclude that this human TES model provides an excellent surrogate for studies of C4.4A and Haldisin expressions in human stratified epithelia.