Change in gene functions (gene cooption) is one of the key mechanisms of molecular evolution. Genes can acquire new functions via alteration in properties of encoded proteins and/or via changes in temporal or spatial regulation of expression. Here we demonstrate radical changes in the functions of orthologous ATP1B4 genes during evolution of vertebrates. Expression of ATP1B4 genes is brain-specific in teleost fishes, whereas it is predominantly muscle-specific in tetrapods. The encoded beta m-proteins in fish, amphibian, and avian species are beta-subunits of Na,K-ATPase located in the plasma membrane. In placental mammals beta m-proteins lost their ancestral functions, accumulate in nuclear membrane of perinatal myocytes, and associate with transcriptional coregulator Ski-interacting protein (SKIP). Through interaction with SKIP, eutherian beta m acquired new functions as exemplified by regulation of TGF-beta-responsive reporters and by augmentation of mRNA levels of Smad7, an inhibitor of TGF-beta signaling. Thus, orthologous vertebrate ATP1B4 genes represent an instance of gene cooption that created fundamental changes in the functional properties of the encoded proteins.