<p>High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern molecule that plays a key role in inflammatory responses and vascular injury. This article elucidates the underlying mechanisms by which HMGB1 regulates the pro-inflammatory phenotypic transformation of vascular endothelial cells (ECs), disrupts vascular barrier homeostasis, bidirectionally regulates angiogenesis, and induces various forms of programmed cell death, such as pyroptosis and ferroptosis, through core receptors, including the receptor for advanced glycation end-products and Toll-like receptors 2 and 4. We also elaborate on how HMGB1 participates in the occurrence and progression of diseases, such as sepsis, pulmonary hypertension, atherosclerosis, ischemia-reperfusion injury, tumors and lung injury, by regulating the biological behaviors of ECs. Although targeting HMGB1 in ECs has significant therapeutic potential, there remains a gap in clinical translation: how to develop novel intervention drugs that can accurately identify the pathogenic conformation of HMGB1 and possess high endothelial targeting capability. This review also summarizes the therapeutic strategies targeting endothelial HMGB1 reported in the literature.</p>