Severe hypoglycemia has a detrimental impact on the cerebrovasculature, but the molecular events that lead to the disruption of the integrity of the tight junctions remain unclear. Here, we report that the microvessel integrity was dramatically compromised (59.41% of wild-type mice) in TP53-induced glycolysis and apoptosis regulator (TIGAR) transgenic mice stressed by hypoglycemia. Melatonin, a potent antioxidant, protects against hypoglycemic stress-induced brain endothelial tight junction injury in the dosage of 400?nmol/L in vitro. FRET (fluorescence resonance energy transfer) imaging data of endothelial cells stressed by low glucose revealed that TIGAR couples with calmodulin to promote TIGAR tyrosine nitration. A tyrosine 92 mutation interferes with the TIGAR-dependent NADPH generation (55.60% decreased) and abolishes its protective effect on tight junctions in human brain microvascular endothelial cells. We further demonstrate that the low-glucose-induced disruption of occludin and Caludin5 as well as activation of autophagy was abrogated by melatonin-mediated blockade of nitrosative stress in vitro. Collectively, we provide information on the detailed molecular mechanisms for the protective actions of melatonin on brain endothelial tight junctions and suggest that this indole has translational potential for severe hypoglycemia-induced neurovascular damage.