High mobility group box 1 (HMGB1) was recently shown to be an important extracellular mediator of severe vascular inflammatory disease, sepsis. Lysozyme (LYZ) has been shown to bind to bacterial lipopolysaccharide (LPS) and have a potential for playing a role in the therapy of inflammatory diseases. However, the effect of LYZ on HMGB1-induced septic response has not been investigated. Moreover, PEGylation effects on the antiseptic activity of LYZ are not known. Here, we show, for the first time, the anti-septic effects of PEGylated LYZ (PEG-LYZ) in HMGB1-mediated inflammatory responses in vitro and in vivo. Among four mono-PEGylated LYZs with different PEGylation sites (N-terminus, Lys(13), Lys(33), and Lys(97)), N-terminally PEGylated LYZ showed the highest activity. Subsequently, among three N-terminally PEGylated LYZs prepared with aldehyde-activated PEGs of 5, 10, and 20 kDa, 5 kDa-PEG-conjugated LYZ (P5-K-1-LYZ) showed the highest antiseptic activity. The data showed that P5-K-1-LYZ post-treatment effectively suppressed LPS-mediated release of HMGB1. P5-K-1-LYZ also inhibited HMGB1-mediated hyperpermeability in human endothelial cells. Furthermore, P5-K-1-LYZ reduced the cecal ligation and puncture (CLP)-induced release of HMGB1 and septic mortality. Collectively, these results suggest P5-K-1-LYZ as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway. (C) 2015 Elsevier Inc. All rights reserved.