Corneal endothelium is responsible for generating an ion flux between the corneal stroma and the anterior chamber of the eye that is necessary for the cornea to remain transparent. However, the ion transport regulatory mechanisms that develop during the formation of the endothelial barrier are not known. In this study, we determined the influence of cell confluence on cell volume and intracellular ionic content on the corneal endothelial cells of rabbits. Our results demonstrate that non-confluent endothelial cells display a hypertrophic volume increase, with higher intracellular contents of potassium and chlorine than those of confluent cells. In contrast, when cells reach confluence and the endothelial barrier forms, cell volume decreases and the intracellular contents of potassium and chlorine decrease. Our genetic analysis showed a higher expression of CFTR and CA2 genes in non-confluent cells, and of the gene KCNC3 in confluent cells. These results suggest that the normal ionic current that keeps the corneal stroma dehydrated and transparent is regulated by cell-cell contacts and endothelial cell confluence, and could explain why the loss of corneal endothelial cells is often associated with corneal edema and even blindness.