Linoleic acid (LA), a dietary unsaturated fatty acid, has been known to increase the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) through the activation of nuclear factor-kappa B. Rho/Rho-kinase (ROCK) pathway mediates various cellular functions related to cardiovascular disease and affects the expression of ICAM-1. However, the exact mechanism underlying this action has not been fully elucidated. In this study, we aimed to find out the role of Rho/ROCK pathway in LA-induced ICAM-1 expression in human aortic endothelial cells (HAECs). We found that LA increased ICAM-1 expression and phosphorylation of ROCK and MYPT-1, a distal signal of ROCK. Y-27632, a ROCK inhibitor, suppressed ICAM-1 expression and phosphorylation of MYPT-1 induced by LA. The effect of LA on the increased phosphorylation of MYPT1 and expression of ICAM-1 was abolished by knocking down RhoA and ROCK2 protein level expression using small interfering RNA. LA increased NF-κB DNA-binding activity, which was inhibited with pretreatment with Y-27632. This study suggests that Rho/ROCK pathway plays a role in LA-induced ICAM-1 expression, which is possibly mediated by NF-κB in HAECs.