Carbon black nanoparticles (CBNs) have been associated with the progression of atherosclerosis. CBNs normally enter the bloodstream and crosslink together to form agglomerates. However, most studies have used nano-sized CB particles to clarify the involvement of CBN exposure in CBN-induced endothelial dysfunction. Herein, we studied endothelial toxicity of CBN aggregates (CBA) to human EA.hy926 vascular cells. Cell viability, lactate dehydrogenase leakage, and oxidative stress were affected by the highest concentration of CBA. Moreover, transmission electron microscopic results showed that CBA entered cells through membrane enclosed vesicles. Rho-associated kinase (ROCK) is involved in regulating vascular diseases. Thus, we co-treated with the of ROCK inhibitor Y-27632 to study whether other adverse effects caused by CBA are related to activating ROCK. As expected, co-treatment with Y-27632 attenuated CBA-induced cytoskeletal damage, dysfunction of the endothelial barrier, and expression of inflammatory factors. Taken together, these results demonstrate that aggregated CBNs can cause endothelial dysfunction possibly by activating ROCK. (C) 2017 Elsevier B.V. All rights reserved.