Three-dimensional (3D) organotypic models are increasingly used to study the aspects of epidermal organisation and cutaneous wound-healing events. However, these are largely dependent on laborious histological analysis and immunohistochemical approaches. Despite the large resource of transgenic and knockout mice harboring mutations relevant to skin disorders, few organotypic mouse skin models are available. We have developed a versatile in vitro 3D organotypic mouse skin equivalent that reflects epidermal organisation in vivo. The system is optically transparent and ideally suited to real-time analysis using a variety of integrated in situ imaging techniques. As a paradigm for coordination of cellular events, the epidermal gap junction network was investigated and the model displayed predominant connexin 43 (Cx43) expression in basal proliferating cells and Cx26 and Cx30 expression in differentiated keratinocytes. We show that attenuation of Cx43-mediated communication by a Cx mimetic peptide enhanced wound closure rates in keratinocyte monocultures and in the living skin equivalent system, emphasising the utility of the model to systematically unravel the molecular mechanisms underlying epidermal morphogenesis, assess promising therapeutic strategies, and reduce animal experimentation. Furthermore, we visualise epidermal regeneration following injury in real time, thereby facilitating avenues to explore distinctive modes of wound re-epithelialisation in a non-invasive manner.