FXYD5 is a Na⁺-K⁺-ATPase regulator, expressed in a variety of normal epithelia. In parallel, it has been found to be associated with several types of cancer and effect lethal outcome by promoting metastasis. However, the molecular mechanism underlying FXYD5 mediated invasion, was has not yet been identified. In this study, using in vivo 4T1 murine breast cancer model, we found that FXYD5 specific shRNA significantly inhibited lung cancer metastasis, without having a substantial effect on primary tumor growth. Our study reveals that FXYD5 participates in multiple stages of metastatic development and exhibits more than one mode of E-cadherin regulation. We provide the first evidence that FXYD5 related morphological changes are mediated through its interaction with Na⁺-K⁺-ATPase. Experiments in cultured 4T1 cells have indicated that FXYD5 expression significantly correlates with down-regulation of the β1 isoform of the pump. This behavior could have implications on both transcellular interactions and intracellular events. Further studies suggest that differential localization of the adaptor protein Annexin A2 in FXYD5 expressing cells may correlate with MMP-9 secretion and adhesion changes in 4T1 WT cells.