Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel -defensin DEFB123. A peptide comprising the -defensin core region was synthesized and used for our analysis. Like other -defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic -defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of -defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.--Motzkus, D., Schulz-Maronde, S., Heitland, A., Schulz, A., Forssmann, W.-G., Jübner, M., and Maronde, E. The novel -defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo.