Nanoparticles (NP) that penetrate external protective barriers of an animal are most likely transported and distributed to various tissues and organs via the circulatory system. Whilst much attention has been dedicated to NP effects on target organs, surprisingly few studies have investigated effects on the vascular system itself, despite the critical role it plays in transporting red and white blood cells, nutrients, as well as coordinating many physiological responses, like immunity and stress. In this study we perfused iliac arteries extracted from cane toads (Rhinella marina) with polyacrylic acid (PAA) coated TiO2 NPs (3-9 nm) to directly assess effects on uptake across luminal endothelial layers, changes to vessel physiological function and toxicity. Perfusion of the iliac artery with 400 µg/mL PAA-TiO2 NPs resulted in histopathological changes in the vascular endothelial layer due to increased endothelial cell death via apoptosis. The PAA-NPs were observed to penetrate at least two cell layers and were present in both endothelial cells and underlying smooth muscle cells. Dual-wire myography experiments revealed that the highest dose of PAA-TiO2 NPs significantly inhibited the ACh-mediated vasodilation in blood vessels by ~50%, which appeared to be attributable to the damaged endothelial layer in the lumen of the vessels. This is the first study to use an ex vivo perfusion method that mimics the blood circulation coupled with dual-wire myography to demonstrate the adverse effects of NPs on blood vessel physiology.