Background: Iron excess is a risk factor for cardiovascular diseases and it is important to understand the effect of iron on vascular permeability, particularly for the transport of large metabolic hormones such as adiponectin. Methods: We used 2-dimensional monolayers of cultured human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) as well as 3-dimensional microvascular networks to measure transendothelial flux. Results: Iron supplementation reduced transendothelial electric resistance (TEER). Flux analysis indicated that under control conditions permeability of 70 kDa dextran and oligomeric forms of adiponectin were restricted in comparison with a 3 kDa dextran, however upon iron treatment permeability of the larger molecules was increased. The increased permeability and size-dependent trans-endothelial movement in response to iron was also observed in 3-dimensional microvascular networks. Mechanistically, the alteration in barrier functionality was associated with increased oxidative stress in response to iron since alterations in TEER and permeability were rescued when reactive oxygen species production was attenuated by pre-treatment with the antioxidant N-acetyl cysteine.]. Conclusions: Iron supplementation induced ROS production resulting in increased transendothelial permeability. General significance: Altogether, this suggests that the oxidative stress associated with iron excess could play an important role in the regulation of endothelial functionality, controlling hormone action in peripheral tissues by regulating the first rate-limiting step controlling hormone access to target tissues.