BACKGROUND: Human cathelicidin LL-37 is a cationic antimicrobial peptide (AMP) which possesses a variety of activities including the ability to neutralise endotoxin. In this study, we investigated the role of LPS neutralisation in mediating LL-37's ability to inhibit Pseudomonas aeruginosa LPS signalling in human monocytic cells. METHODOLOGY/PRINCIPAL FINDINGS: Pre-treatment of monocytes with LL-37 significantly inhibited LPS-induced IL-8 production and the signalling pathway of associated transcription factors such as NF-κB. However, upon removal of LL-37 from the media prior to LPS stimulation, these inhibitory effects were abolished. These findings suggest that the ability of LL-37 to inhibit LPS signalling is largely dependent on extracellular LPS neutralisation. In addition, LL-37 potently inhibited cytokine production induced by LPS extracted from P. aeruginosa isolated from the lungs of cystic fibrosis (CF) patients. In the CF lung, polyanionic molecules such as glycosaminoglycans (GAGs) and DNA bind LL-37 and impact negatively on its antibacterial activity. In order to determine whether such interactions interfere with the LPS neutralising ability of LL-37, the status of LL-37 and its ability to bind LPS in CF sputum were investigated. Overall our findings suggest that in the CF lung, the ability of LL-37 to bind LPS and inhibit LPS-induced IL-8 production is attenuated as a result of binding to DNA and GAGs. However, LL-37 levels and its concomitant LPS-binding activity can be increased with a combination of DNase and GAG lyase (heparinase II) treatment. CONCLUSIONS/SIGNIFICANCE: Overall, these findings suggest that a deficiency in available LL-37 in the CF lung may contribute to greater LPS-induced inflammation during CF lung disease.