Our previous studies demonstrated that high mobility group box‐1 (HMGB1), a typical damage‐associated molecular pattern (DAMP) protein, is associated with the disease activity of antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). Moreover, HMGB1 participates in ANCA‐induced neutrophil activation. The current study aimed to investigate whether HMGB1 regulated the interaction between neutrophils and glomerular endothelial cells (GEnC) in the presence of ANCA. Correlation analysis on HMGB1 levels in AAV patients and soluble intercellular cell adhesion molecule‐1 (sICAM‐1) levels or vascular endothelial growth factor (VEGF) levels, which are markers of endothelial cell activation, was performed. The effect of HMGB1 on neutrophil migration towards GEnC, respiratory burst and degranulation of neutrophils in coculture conditions with GEnC was measured. The activation of neutrophils, the activation and injury of GEnC, and the consequent pathogenic role of injured GEnC were evaluated. Plasma levels of HMGB1 correlated with sICAM‐1 and VEGF (r = 0.73, P < 0.01; r = 0.41, P = 0.04) in AAV patients. HMGB1 increased neutrophil migration towards GEnC, as well as respiratory burst and degranulation of neutrophils in the presence of ANCA in the coculture system. In the presence of robust neutrophil activation, GEnC were further activated and injured in the coculture system of GEnC and neutrophils. In addition, injured GEnC could produce TF‐positive leuco‐endothelial microparticles and endothelin‐1 (ET‐1), while NF‐κB was phosphorylated (S529) in the injured GEnC. Plasma levels of HMGB1 correlated with endothelial cell activation in AAV patients. HMGB1 amplified neutrophil activation and the activation and injury of GEnC in the presence of ANCA.