Abstract Body: Diabetes mellitus (DM) significantly increases the risk of microvascular dysfunction, which can lead to complications such as nephropathy, cardiomyopathy, retinopathy, and peripheral vascular disease. This dysfunction is largely driven by persistent hyperglycemia, resulting in endothelial mitochondrial dysfunction. High glucose conditions increase mitochondrial superoxide production and reduce endothelial-derived nitric oxide (NO) bioavailability, impairing vasodilation and promoting vascular inflammation. Mitochondrial Fission Protein-1 (Fis1), known for its role in mitochondrial fission, also regulates mitophagy, a process critical for maintaining mitochondrial health. Elevated Fis1 expression has been observed in the diabetic endothelium and is associated with impaired endothelial function, though its precise mechanism remains unclear. We hypothesize that Fis1 overexpression in DM inhibits mitophagy, leading to reduced NO bioavailability, increasing mitochondrial superoxide production, and impairing vasoreactivity. Using human umbilical vein endothelial cells (HUVECs), we investigated mitophagic flux, NO bioavailability, and superoxide production under high glucose conditions. Endothelial-specific Fis1 overexpression reduced NO levels (N=3, p<0.05), while siRNA-mediated knockdown of Fis1 rescued NO bioavailability (N=3, p<0.01). High glucose increased superoxide production (N=2, p<0.01), which was reversed by Fis1 knockdown or mitophagy activation with T271 (N=2, p<0.01). Mitophagy, measured using mitoSRAI (a live cell mitophagy indicator), was inhibited under high glucose but enhanced by Fis1 knockdown or inhibition with Fis1 inhibitor pep213 (N=5, p<0.01), reducing mitochondrial fragmentation. This study reveals a novel role for Fis1 in diabetic endothelial dysfunction through its regulation of mitophagy. Targeting Fis1 to restore mitophagy may improve endothelial health and microvascular function in DM, offering a potential therapeutic strategy.