Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia-mediated barrier dysfunctions. Dyn-2 (dynamin-2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn-2 in hypoxia-mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn-2 by transfecting the cells with K44A, a dominant negative construct of dyn-2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia-mediated leakiness of K44A-overexpressed endothelial monolayer. Our study confirms that inhibition of dyn-2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia.