FXYD5, also known as Dysadherin, belongs to a family of tissue-specific regulators of the Na,K-ATPase. We determined the kinetics effects of FXYD5 on Na,K-ATPase pump activity in stably transfected MDCK cells. FXYD5 significantly increased the apparent affinity for Na⁺ 2-fold, and decreased the apparent affinity for K⁺ by 60% with a 2-fold increase in V_max(K+), a pattern that would increase activity and Na⁺ removal from the cell. To test the effect of increased sodium uptake on FXYD5 expression, we analyzed MDCK cells stably transfected with an inducible vector expressing all three subunits of the epithelial sodium channel (ENaC). Na,K-ATPase activity increased 6-fold after 48-hour ENaC induction, but FXYD5 expression decreased 75%. FXYD5 expression was also decreased in lung epithelia from mice that overexpress ENaC, suggesting that chronic Na⁺ absorption by itself downregulates epithelial FXYD5 expression. Patients with cystic fibrosis (CF) display ENaC-mediated hyperabsorption of Na⁺ in the airways, accompanied by increased Na,K-ATPase activity. However, FXYD5 was significantly increased in the lungs and nasal epithelium of CF mice as assessed by RT-PCR, immunohistochemistry and immunoblot analysis (P<0.001). FXYD5 was also upregulated in nasal scrapings from human CF patients compared to controls (P<0.02). Treatment of human tracheal epithelial (HTE) cells with a CFTR inhibitor (I-172) confirmed that loss of CFTR function correlated with increased FXYD5 expression (P<0.001), which was abrogated by an inhibitor of NF-B. Thus FXYD5 is upregulated in CF epithelia and this change may exacerbate the Na⁺ hyperabsorption and surface liquid dehydration observed in CF airway epithelia.