Skin is the main component of the vertebrate integumentary system and its primary function is to act as a protective barrier. Due to its crucial role as a barrier tissue, the basic structure of vertebrate skin (e.g. epidermis, dermis) can be observed across species. By separating internal and external environments, skin can help to prevent pathogen invasion, regulate body temperature, and maintain electrolyte and fluid balance. Teleost fishes do not possess heavily keratinized outer layers of skin. Instead, the skin of these fishes presents an outer layer of living epithelial cells that are in direct contact with the water and must deal directly with the challenges of life in an aquatic setting. For example, in freshwater (FW), teleosts possess hyperosmotic internal fluids, which leads to passive ion loss across tissues that interface with the surroundings. Yet despite a complex structure and extensive contact with the external environment, the skin of adult FW fishes is classically regarded as a static, passive barrier to diffusional ion loss. Recent studies have shown that select tight junction (TJ) proteins alter in transcript abundance during salinity acclimation, but a broader picture of how the molecular components of the fish skin TJ complex respond to environmental change is lacking. The present study examined regional TJ protein response to environmental change (i.e. acclimation to ion-poor water, IPW) in FW rainbow trout (Oncorhynchus mykiss) skin. Acute and chronic treatment with IPW resulted in alterations of Cldn protein abundance in a region-specific and time-dependent manner. This study also provided a new look into an ex vivo technique, the Franz Cell, for measuring ion flux and paracellular permeability of fish skin. These data provide a unique look at the region-specific changes of TJ complex components in the adult fish skin and connect them to region-specific permeability properties.