As a late mediator of inflammation, the high-mobility group box 1 protein (HMGB1) plays a key role in the inflammatory responses to tissue injury and infection by inducing and extending the production of proinflammatory cytokines. It has been observed that lower concentration thrombin mediates anti-inflammatory activities. The aim of this study was to investigate whether lower concentration thrombin could modulate HMGB1 expression and could inhibit HMGB1-mediated inflammatory responses in human umbilical vein endothelial cells (HUVECs). Here, results showed that lower concentration thrombin or thrombin receptor agonist peptide inhibits lipopolysaccharide-induced HMGB1 release from HUVECs. And lower concentration thrombin has inhibitory effects not only on the expression of cell adhesion molecules but also on neutrophils adhesion and migration toward HUVECs in response to HMGB1. Interestingly, the HMGB1-induced nuclear factor kappa B activation and tumor necrosis factor-alpha release from HUVECs were inhibited by lower concentration thrombin. Given these results, lower concentration thrombin could be a strong candidate as a therapeutic agent for various systemic inflammatory diseases.