The Na,K-ATPase, consisting of two essential subunits (alpha, beta), plays a critical role in the regulation of ion homeostasis in mammalian cells. Recent studies indicate that reduced expression of the beta1 isoform (NaK-beta1) is commonly observed in carcinoma and is associated with events involved in cancer progression. In this study, we present evidence that repletion of NaK-beta1 in Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK), a highly tumorigenic cell line, inhibits anchorage independent growth and suppresses tumor formation in immunocompromised mice. Additionally, using an in vitro cell-cell aggregation assay, we showed that cell aggregates of NaK-beta1 subunit expressing MSV-MDCK cells have reduced extracellular regulated kinase (ERK) 1/2 activity compared with parental MSV-MDCK cells. Finally, using immunohistochemistry and fully quantitative image analysis approaches, we showed that the levels of phosphorylated ERK 1/2 are inversely correlated to the NaK-beta1 levels in the tumors. These findings reveal for the first time that NaK-beta1 has a potential tumor-suppressor function in epithelial cells.