Freshwater (FW) fishes reside exist in aquatic habitats that are more dilute than their inner fluids. This leads to passive ion loss across tissues that directly interface with the surrounding medium, such as the gill and skin. The barrier properties of adult teleost fish skin in FW are well documented and in most cases, the trunk skin of adult fishes is void of any significant capacity for active ion movement. Therefore, despite its complex structure and extensive contact with the external environment, the skin of adult fishes is classically regarded as a relatively static, passive barrier to diffusional ion loss in FW. Nevertheless, recent observations have revealed the presence of a considerable number of genes encoding tight junction (TJ) proteins in the adult skin of several fish species. In addition, some studies have also shown that select TJ proteins in the skin of fish significantly alter in abundance upon exposure to environments that vary in ionic composition. This suggests that the molecular architecture of the skin TJ complex may play an important role in regulating skin permeability during environmental change. Despite this, almost nothing is known about how the molecular physiology of the TJ complex in this boundary tissue is regulated. Additionally, structural and functional differences are known to exist along the dorsoventral axis of the adult fish skin. However, none of the studies conducted to date, have taken this documented regional heterogeneity into account. This study examined TJ protein response to environmental change (acclimation to ion‐poor water, IPW) in freshwater (FW) rainbow trout (Oncorhynchus mykiss) skin. Changes in TJ protein abundance along the dorsoventral axis of the trout skin taken into consideration, as were possible roles for prolactin and cortisol, hormones involved in salinity‐dependent regulation of TJ/passive permeability. Differences in abundance of cldn‐3a, −5b, −6, −10c, −30, −32a and zo‐1 were detected along the dorsoventral axis of rainbow trout skin and many of these were found to be enriched in the lateral and dorsolateral regions. In addition, cldn mRNA abundance altered in response to IPW acclimation in a region‐specific manner. Osmoregulatory hormones, cortisol and prolactin, and their receptors were implicated in the regulation of Cldn transcript abundance in rainbow trout skin. These data provide a unique look at the region‐specific endocrine regulation of TJ complex components in adult fish skin.Support or Funding InformationThis work was funded by National Science and Engineering Research Council of Canada