Antioxidants have an important role in protecting against diabetes complications such as vascular endothelial cell damage. Fucoidan has strong antioxidant properties, therefore the aim of this study was to investigate the protective mechanism of fucoidan nanoparticles through the pathway of antioxidant activity against streptozotocin-induced diabetic aortic endothelial cell dysfunction in rats. Fucoidan nanoparticles are made utilizing high-energy ball milling. This research consists of five groups, namely: control rats, rats were administered aquadest; diabetic rats, rats were administered streptozotocin (STZ); fucoidan nanoparticle rats, rats were administered STZ and fucoidan nanoparticles. Aortic tissue was collected for the evaluation of ROS (reactive oxygen species), Malondialdehyde (MDA), superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), Nuclear factor erythroid-2-related factor 2 (Nrf2), Nitric Oxide (NO), cyclic Guanosine Monophosphate (cGMP), relaxation response of acetylcholine (Ach), and the diameter of the aorta. The size distribution of the fucoidan nanoparticles was 267.2 ± 42.8 nm. Administration of fucoidan nanoparticles decreased the levels of ROS and MDA, and increased the levels of SOD, levels of GPx, Nrf2 expression, NO levels, cGMP expression, the relaxation response of Ach, and lumen diameter of the aorta, which are significantly different when compared with diabetic rats, p < 0.05. In this study, we concluded that the mechanism pathway of fucoidan nanoparticles prevents aortic endothelial cell dysfunction in diabetic rats through antioxidant activity by reducing ROS and MDA and incrementing SOD levels, GPx levels, and Nrf2 expression. All of these can lead to an elevated relaxation response effect of Ach and an increase in the lumen diameter of the aorta, which indicates a protective effect of fucoidan nanoparticles on aortic endothelial cells.