FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1?β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1?β1 interactions, suggesting that the ratio between FXYD5 and α1?β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion. Fil: Tokhtaeva, Elmira. University of California at Los Angeles; Estados Unidos Fil: Sun, Haying. Northwestern University; Estados Unidos Fil: Deiss Yehiely, Nimrod. Northwestern University; Estados Unidos Fil: Wen, Yi. University of California at Los Angeles; Estados Unidos Fil: Soni, Pritin N.. Northwestern University; Estados Unidos Fil: Gabrielli, Nieves María. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Northwestern University; Estados Unidos Fil: Marcus, Elizabeth A.. University of California at Los Angeles; Estados Unidos Fil: Ridge, Karen M.. Northwestern University; Estados Unidos Fil: Sachs, George. University of California at Los Angeles; Estados Unidos Fil: Vazquez, Monica Hebe. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Sznajder, Jacob I.. Northwestern University; Estados Unidos Fil: Vagin, Olga. University of California at Los Angeles; Estados Unidos Fil: Dada, Laura Andrea. Northwestern University; Estados Unidos