Recently, corticosteroid hormone-induced factor (CHIF) and the γ-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase α1–β1, α2–β1 and α3–β1 but not with α–β2 isozymes, whereas, in brain, it is only associated with α1–β isozymes. FXYD7 decreases the apparent K+ affinity of α1–β1 and α2–β1, but not of α3–β1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.