The microvasculature comprises blood vessels (<200 μm in diameter) that nourish and drain parenchymal and stromal cells throughout the body. These vessels are lined by a single-cell layer of endothelial cells (ECs) that share common features while exhibiting differing characteristics across tissues and vascular segments within the same tissue. In homeostasis, microvascular ECs actively regulate blood flow, maintain blood fluidity, control water, solute, and macromolecular transfer between blood and tissues, and modulate circulating immune cell recruitment and activation. Advances in single-cell transcriptional profiling have provided profound insights into the differences between ECs from specific organs and even within microvascular segments (i.e., arterioles, capillaries, and venules). This specialization of ECs in health reflects the broad diversity of capillary function to match the demands of specialized tissues. In disease, microvascular ECs may be activated by both paracrine and systemic factors to regulate blood flow, coagulation, permselectivity, and immune cell responses. This activation can occur in rapid (type I), acute (type II), and chronic (type III) phases, all of which aim to restore homeostasis. Endothelial dysfunction occurs when these processes become exaggerated or break down, thereby propagating pathology and potentially resulting in endothelial cell death. Recent profiling of microvascular ECs in health and disease has both deepened prior insights into pathological processes and identified new targets for clinical investigation.