S100 calcium binding protein B (S100B) is recently known as the markers for inflammatory diseases. However, its roles and underlying mechanism on multiple-traumas remain unclearly. In this study, total 123 patients (87 male and 36 female) were enrolled and divided into two group: Injury severity score (ISS) ≥ 16 (n = 69); ISS < 16 (n = 54). ELISA assay confirmed that the circulating S100B levels in multi-trauma were obviously higher than that in healthy volunteers. Additionally, S100B concentrations was associated with injury severity as an obviously higher levels of S100B (2.18 μg/L) in severe trauma group (ISS ≥ 16) than 1.26 μg/L in moderate trauma group (ISS < 16). Furthermore, the average concentration of S100B was 2.91 μg/L (n = 14) in fatal patients and 2.21 μg/L in survivors, suggesting an obvious correlation between S100B and the severity degree of multi-injury. Further analysis confirmed an obvious correlation between S100B levels and sE-selectin, and Von willebrand factor (vWF), both of these are the marker of endothelial cell injury. After transfection with pcDNA3.1-S100B, human umbilical vein endothelial cells (HUVECs) cell apoptotic ratio was dramatically up-regulated, concomitant with the increase in IL-6 and IL-8 levels, suggesting that S100B might regulate the development of polytrauma by mediating endothelial cell dysfunction. Together, these results suggest a potential predictive value of S100B and its underlying mechanism in the pathological process of polytrauma. Therefore, this study will support the potential clinical aspect for the diagnostic and treatment of polytrauma and its complications.