Abstract Body (Do not enter title and authors here): Diabetic vascular dysfunction is a serious health concern, affecting ~38 million people in the USA, according to the 2020 CDC-National Diabetes Statistics Reports. Type 2 diabetes (T2D) causes several vascular changes leading to major clinical consequences such as stroke and ~3-fold elevated risk of cardiovascular diseases (CVD) and the underlying causes of vascular dysfunction in T2D are complex. The anatomical position of endothelial cells (ECs) between the circulating blood and vessel walls makes them the first to encounter and respond to cellular stressors in the bloodstream. Here, we investigated the role of long-term increases in plasma histamine levels as observed in T2D and its effects on ECs and vascular contractility. The high-fat diet-low-dose streptozotocin protocol induced T2D in C57BL/6J mice. Endothelial cell function was analyzed using Western blotting, in vitro blood-brain barrier assays, histamine measurements, immunofluorescence, and pressurized artery myography. In our T2D model, EC-mediated histamine synthesis occurred after the onset of IR. A spike in plasma histamine levels was observed immediately following the onset of insulin resistance, followed by a brief plateau. A second spike in plasma histamine levels was noted four weeks later. In isolated control ECs, the addition of external histamine showed a concentration-dependent increase in histidine decarboxylase expression, and cellular histamine levels were observed, which was inhibited by the H2R blocker, famotidine. The tight junction marker, JAM-A (CD321), expression in diabetic cerebral arteries was ~42% less compared to the non-diabetic controls. Transendothelial Electrical Resistance testing showed a ~2-fold higher permeability with diabetic ECs than controls. Pressure myography on isolated cerebral arteries indicated that external histamine passed through the lumen of arteries from T2D mice 4 weeks after onset of IR constricted whereas control arteries dilated. Additionally, T2D mice cerebral artery myogenic tone at 60 mmHg was 70% greater compared to control arteries. These data indicate that the onset of IR triggers an increase in circulating histamine levels, which in turn stimulates histamine synthesis and release from endothelial cells leading to the loss of endothelial barrier function, blood-brain barrier leakage, and vasoconstriction. We are currently investigating the molecular pathways that precede the increase in circulating histamine levels during T2D.