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Arteriosclerosis is the major cause of coronary artery and cerebrovascular disease, which are leading causes of death. Pro-inflammatory cytokines induce injury to vascular endothelial cells by increasing cell adhesion molecules, leading to vascular inflammation, a major risk factor for the development of arteriosclerosis. In the current study, we investigated the inhibitory effect of enzymatic hydrolysate from Japanese mud shrimp Upogebia major on the inflammation of tumor necrosis factor-α (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). We first evaluated the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of eight U. major enzymatic hydrolysates: alcalase, papain, α-chymotrypsin (α-Chy), trypsin, pepsin, neutrase, protamex and flavourzyme. Of these, α-Chy exhibited potent antioxidant and ACE inhibitory activities. The α-Chy hydrolysate was fractionated by two ultrafiltration membranes of 3 and 10 kDa. The α-Chy hydrolysate of U. major and its molecular weight cut-off fractions resulted in a significant reduction in NO production and a decrease in cell adhesion molecules [vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and endothelial-selectin (E-selectin)] and pro-inflammatory cytokines [interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1)] in TNF-α-stimulated HUVECs. These results suggest that enzymatic hydrolysate from U. major can be used in the control and prevention of vascular inflammation and arteriosclerosis.