Significance Statement: The specialized vessels comprising the renal vasculature are characterized by highly differentiated renal endothelial cell types, but this heterogeneity has been poorly inventoried. Using single-cell RNA sequencing, the authors developed a high-resolution atlas of mouse renal endothelial cells. They also investigated how medullary renal endothelial cells adapt to a switch from diuresis to antidiuresis. This study describes the molecular and metabolic adaptation of medullary renal endothelial cells to dehydration, and uncovers a role for mitochondrial oxidative phosphorylation in hyperosmolarity conditions to allow for urine concentration. The authors’ atlas of mouse renal endothelial cells provides a resource for future studies, and their findings may provide insights into cardiometabolic or kidney diseases involving hyperosmolarity and dehydration, in which urine concentration capacity is perturbed. Background: Renal endothelial cells from glomerular, cortical, and medullary kidney compartments are exposed to different microenvironmental conditions and support specific kidney processes. However, the heterogeneous phenotypes of these cells remain incompletely inventoried. Osmotic homeostasis is vitally important for regulating cell volume and function, and in mammals, osmotic equilibrium is regulated through the countercurrent system in the renal medulla, where water exchange through endothelium occurs against an osmotic pressure gradient. Dehydration exposes medullary renal endothelial cells to extreme hyperosmolarity, and how these cells adapt to and survive in this hypertonic milieu is unknown. Methods: We inventoried renal endothelial cell heterogeneity by single-cell RNA sequencing >40,000 mouse renal endothelial cells, and studied transcriptome changes during osmotic adaptation upon water deprivation. We validated our findings by immunostaining and functionally by targeting oxidative phosphorylation in a hyperosmolarity model in vitro and in dehydrated mice in vivo. Results: We identified 24 renal endothelial cell phenotypes (of which eight were novel), highlighting extensive heterogeneity of these cells between and within the cortex, glomeruli, and medulla. In response to dehydration and hypertonicity, medullary renal endothelial cells upregulated the expression of genes involved in the hypoxia response, glycolysis, and—surprisingly—oxidative phosphorylation. Endothelial cells increased oxygen consumption when exposed to hyperosmolarity, whereas blocking oxidative phosphorylation compromised endothelial cell viability during hyperosmotic stress and impaired urine concentration during dehydration. Conclusions: This study provides a high-resolution atlas of the renal endothelium and highlights extensive renal endothelial cell phenotypic heterogeneity, as well as a previously unrecognized role of oxidative phosphorylation in the metabolic adaptation of medullary renal endothelial cells to water deprivation.