Hyaluronan (HA) is a major extracellular matrix component of the skin. Amongst its biological functions is the maintenance of epidermal homeostasis. The mechanisms of action, however, remain unclear. To explore the interaction of HA with the epidermis, we have looked at the effects of exogenous application of HA in an organotypic culture model containing a dermal substrate with and without fibroblast incorporation. The results demonstrate that exogenous HA enhances epidermal proliferation resulting in a thicker viable epidermis with an increase in the number and intensity of Ki67-positive basal cells; HA also improves the basement membrane assembly as evidenced by an increased expression of laminin-332 and collagen type IV but not the expression of Nidogen-1 at the epidermal-dermal junctional zone; furthermore, the development of epidermal lipid barrier structure was enhanced. These findings provide evidence to support the therapeutic use of exogenous HA for treating skin disorders with aberrant epidermal homeostasis.