Background Endothelial glycocalyx breakdown elicits syndecan-1 shedding and endotheliopathy of trauma (EoT). We hypothesized that a cutoff syndecan-1 level can identify patients with endothelial dysfunction who would have poorer outcomes. Study Design We conducted a prospective observational study. Trauma patients with the highest level of activation admitted from July 2011 through September 2013 were eligible. We recorded demographics, injury type/severity (Injury Severity Score), physiology and outcomes data, and quantified syndecan-1 and soluble thrombomodulin from plasma with ELISAs. With receiver operating characteristic curve analysis, we defined EoT+ as the syndecan-1 cutoff level that maximized the sum of sensitivity and specificity (Youden index) in predicting 24-hour in-hospital mortality. We stratified by this cutoff and compared both groups. Factors associated with 30-day in-hospital mortality were assessed with multivariable logistic regression (adjusted odds ratios and 95% CIs reported). Results From receiver operating characteristic curve analysis (area under the curve = 0.71; 95% CI 0.58 to 0.84), we defined EoT+ as syndecan-1 level ≥40 ng/mL (sensitivity = 0.62, specificity = 0.73). Of the 410 patients evaluated, 34% (n = 138) were EoT+ patients, who presented with higher Injury Severity Scores (p < 0.001) and blunt trauma frequency (p = 0.016) than EoT− patients. Although EoT+ patients had lower systolic blood pressure (median 119 vs 128 mmHg; p < 0.001), base excess and hemoglobin were similar between groups. The proportion of transfused (EoT+ 71.7% vs EoT− 36.4%; p < 0.001) and deceased EoT+ patients (EoT+ 24.6% vs EoT− 12.1%; p < 0.001) was higher. EoT+ was significantly associated with 30-day in-hospital mortality (adjusted odds ratio = 2.23; 95% CI 1.22 to 4.04). Conclusions A syndecan-1 level ≥40 ng/mL identified patients with significantly worse outcomes, despite admission physiology similar to those without the condition.