The amount of Na+ and K+ in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of Na+ and K+ in the regulation of two pleckstrin homology domain-containing intracellular signaling molecules, phospholipase C (PLC)-γ1 and epithelial and endothelial tyrosine kinase/bone marrow tyrosine kinase on chromosome X (Bmx), and agonist-generated Ca2+ signaling in the endothelium. Extracellular K+ concentration regulated the levels of activated PLC-γ1, Bmx, and carbachol-stimulated intracellular Ca2+ mobilization in human endothelial cells. Additional experiments confirmed that high-conductance Ca2+-activated K+ channels and phosphatidylinositol 3-kinase mediated these effects. The content of Na+and K+ in the diet also regulated Bmx levels in endothelial cells and activated PLC-γ1 levels in rats in vivo. The effects of dietary K+ on Bmx were more pronounced in rats fed a high-salt diet compared with rats fed a low-salt diet. These experiments elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary Na+ and K+ content. © 2014 the American Physiological Society.