Background: Carbon nanotubes (CNTs) have many potential applications, including as delivery systems for a variety of diagnostic or therapeutic agents. However, it has been suggested that exposure to carbon nano-materials may be a risk for the development of vascular diseases due to its impact on the vascular endothelium. Materials and Methods: Male Sprague-Dawley rats (180–200 g) were used to generate an atherosclerosis (AS) model, and the effect of intravenous administration of multi-walled carbon nanotubes (MWCNTs) on AS was studied. To further understand the underlying mechanisms, the effects of exposure of human umbilical vascular endothelial cells (HUVECs) to MWCNTs were examined. Results: Exposure to 200 μg/kg MWCNTs aggravated AS in this model. In addition, exposure to 50, 100 and 200 μg/kg MWCNTs increased the calcification of the aorta in the model. Short-term exposure also revealed that 200 μg/kg MWCNTs injured the endothelium in the aorta. MWCNTs disrupted the endothelial tight junction and induced endothelial cell death. Conclusion: The results demonstrated that MWCNTs could induce structural and functional changes in the endothelium, probably through vascular endotheliocyte injury, which eventually affected the development of AS in SD rats.