The Na,K-ATPase, consisting of α- and β-subunits, regulates intracellular ion homeostasis. Recent studies have demonstrated that Na,K-ATPase also regulates epithelial cell tight junction structure and functions. Consistent with an important role in the regulation of epithelial cell structure, both Na,K-ATPase enzyme activity and subunit levels are altered in carcinoma. Previously, we have shown that repletion of Na,K-ATPase β₁-subunit (Na,K-β) in highly motile Moloney sarcoma virus-transformed Madin-Darby canine kidney (MSV-MDCK) cells suppressed their motility_. However, until now, the mechanism by which Na,K-β reduces cell motility remained elusive. Here, we demonstrate that Na,K-β localizes to lamellipodia and suppresses cell motility by a novel signaling mechanism involving a cross-talk between Na,K-ATPase α₁-subunit (Na,K-α) and Na,K-β with proteins involved in phosphatidylinositol 3-kinase (PI3-kinase) signaling pathway. We show that Na,K-α associates with the regulatory subunit of PI3-kinase and Na,K-β binds to annexin II. These molecular interactions locally activate PI3-kinase at the lamellipodia and suppress cell motility in MSV-MDCK cells, independent of Na,K-ATPase ion transport activity. Thus, these results demonstrate a new role for Na,K-ATPase in regulating carcinoma cell motility.