The pro-inflammatory chemokine interleukin-8 (CXCL8) exerts its function by establishing a chemotactic gradient in infected or damaged tissues to guide neutrophil granulocytes to the site of inflammation via its G protein-coupled receptors (GPCRs) CXCR1 and CXCR2 located on neutrophils. Endothelial glycosaminoglycans (GAGs) have been proposed to support the chemotactic gradient formation and thus the inflammatory response by presenting the chemokine to approaching leukocytes. In this study, we show that neutrophil transmigration in vitro can be reduced by adding soluble GAGs and that this process is specific with respect to the nature of the glycan. To further investigate the GAG influence on neutrophil migration, we have used an engineered CXCL8 mutant protein (termed PA401) which exhibits a much higher affinity towards GAGs and an impaired GPCR activity. This dominant-negative mutant chemokine showed anti-inflammatory activity in various animal models of neutrophil-driven inflammation, i.e. in urinary tract infection, bleomycin-induced lung fibrosis, and experimental autoimmune uveitis. In all cases, treatment with PA401 resulted in a strong reduction of transmigrated inflammatory cells which became evident from histology sections and bronchoalveolar lavage. Since our CXCL8-based decoy targets GAGs and not GPCRs, our results show for the first time the crucial involvement of this glycan class in CXCL8/neutrophil-mediated inflammation and will thus pave the way to novel approaches of anti-inflammatory treatment.