Disruption of the endothelial glycocalyx has a prominent role in the pathophysiology of sepsis. Developmental endothelial locus-1 (Del-1) is an endothelial-derived anti-inflammatory factor. We hypothesized that degradation of the endothelial glycocalyx during sepsis may increase serum Del-1. A mouse model of sepsis was created using cecal ligation and puncture. In septic mice, the endothelial glycocalyx was nearly completely degraded, with less formation of Del-1 in the endothelium and extracellular matrix than in control mice. Serum Del-1 levels were significantly increased in the septic mice with increasing severity of sepsis. Serum Del-1 levels were also measured in 84 patients with sepsis and septic shock and in 20 control subjects. The median serum Del-1 level in patients with sepsis was significantly higher than that in healthy controls. The high Del-1 group had higher illness severity scores and contained more patients with organ dysfunction than the low Del-1 group. The 90-day mortality rate was significantly higher in the high Del-1 group than in the low Del-1 group. Multivariate analysis indicated a tendency for a high serum Del-1 level to be associated with a higher mortality risk. Increased serum Del-1 may be a novel diagnostic biomarker of sepsis and an indicator of disease severity.