Human apolipoprotein A-I (apoA-I) mimetic L-4F inhibits acute inflammation in endotoxemic animals. Since neutrophils play a crucial role in septic inflammation, we examined the effects of L-4F, compared to apoA-I, on lipopolysaccharide (LPS)-mediated activation of human neutrophils. We performed bioassays in human blood, isolated human neutrophils (incubated in 50 % donor plasma), and isolated human leukocytes (incubated in 5 and 50 % plasma) in vitro. In whole blood, both L-4F and apoA-I inhibited LPS-mediated elevation of TNF-α and IL-6. In LPS-stimulated neutrophils, L-4F and apoA-I (40 μg/ml) also decreased myeloperoxidase and TNF-α levels; however, L-4F tended to be superior in inhibiting LPS-mediated increase in IL-6 levels, membrane lipid rafts abundance and CD11b expression. In parallel experiments, when TNF-α and IL-8, instead of LPS, was used for cell stimulation, L-4F and/or apoA-I revealed only limited efficacy. In LPS-stimulated leukocytes, L-4F was as effective as apoA-I in reducing superoxide formation in 50 % donor plasma, and more effective in 5 % donor plasma (P < 0.05). Limulus ambocyte lysate (LAL) and surface plasmon resonance assays showed that L-4F neutralizes LAL endotoxin activity more effectively than apoA-I (P < 0.05) likely due to avid binding to LPS. We conclude that (1) direct binding/neutralization of LPS is a major mechanism of L-4F in vitro; (2) while L-4F has similar efficacy to apoA-I in anti-endotoxin effects in whole blood, it demonstrates superior efficacy to apoA-I in aqueous solutions and fluids with limited plasma components. This study rationalizes the utility of L-4F in the treatment of inflammation that is mediated by endotoxin-activated neutrophils.