Cardiotonic steroids signaling through the basolateral sodium pump (Na/K-ATPase) have been shown to alter renal salt handling in intact animals. Because the relationship between renal salt handling and blood pressure is a key determinant of hypertension, and patients with insulin resistance are frequently hypertensive, we chose to examine whether there might be competition for resources necessary for receptor-mediated endocytosis. In LLC-PK1 cells, the Na/K-ATPase-α1 and carcinoembryonic antigen cell adhesion molecule 1, a plasma membrane protein that promotes receptor-mediated endocytosis, colocalized in the plasma membranes and translocated to the intracellular region in response to ouabain. Either ouabain or insulin alone caused accumulation of and carcinoembryonic antigen cell adhesion molecule, as well as insulin receptor-β, and epidermal growth factor receptor in early endosomes, but no synergy was demonstrable. Like ouabain, insulin also caused c-Src activation. When caveolin or Na/K-ATPase-α1 expression was knocked down with small interfering RNA, insulin but not ouabain induced carcinoembryonic antigen cell adhesion molecule 1, insulin receptor-β, and epidermal growth factor receptor endocytosis. To determine whether this might be relevant to salt handling in vivo, we examined salt loading in mice with null renal carcinoembryonic antigen cell adhesion molecule 2 expression. The null renal carcinoembryonic antigen cell adhesion molecule 2 animals demonstrated greater increases in blood pressure with increases in dietary salt than control animals. These data demonstrate that cardiotonic steroids and insulin compete for cellular endocytosis resources and suggest that, under conditions where circulating insulin concentrations are high, cardiotonic steroid-mediated natriuresis could be impaired.