Abstract: The recently defined FXYD protein family contains seven members that are small, single‐span membrane proteins characterized by a signature sequence containing an FXYD motif and three other conserved amino acid residues. Until recently, the functional role of FXYD proteins was largely unknown, with the exception of the γ subunit of Na,K‐ATPase, which was shown to be a specific regulator of renal α1‐β1 isozymes. We have investigated whether other members of the FXYD family may have a similar role as the γ subunit and have found that CHIF (corticosteroid hormone‐induced factor, FXYD4), FXYD7, as well as phospholemman (FXYD1) specifically associate with Na,K‐ATPase and preferentially with α1‐β isozymes in native tissues, and produce distinct effects on the transport properties of Na,K‐ATPase that are adapted to the physiological demands of the tissues in which they are expressed. These results provide evidence for a unique and novel mode of regulation of Na,K‐ATPase by FXYD proteins that involves a tissue‐specific expression of an auxiliary subunit of distinct Na,K‐ATPase isozymes.