Endotoxemia is a major cause of chronic inflammation, and is an important pathogenic factor in the development of metabolic syndrome and atherosclerosis. Human apolipoprotein E (apoE) and apoA-I are protein components of high-density lipoprotein, which have strong anti-endotoxin activity. Here, we compared anti-endotoxin activity of Ac-hE18A-NH2 and 4F peptides, modified from model amphipathic helical 18A peptide, to mimic, respectively, apoE and apoA-I properties. Ac-hE18A-NH2, stronger than 4F, inhibited endotoxin activity and disaggregated Escherichia coli 055:B5 (wild smooth serotype). Ac-hE18A-NH2 and 4F inhibited endotoxin activity of E. coli 026:B6 (rough-like serotype) to a similar degree. This suggests that Ac-hE18A-NH2 as a dual-domain molecule might interact with both the lipid A and headgroup of smooth LPS, whereas 4F binds lipid A. In C57BL/6 mice, Ac-hE18A-NH2 was superior to 4F in inhibiting the inflammatory responses mediated by E. coli 055:B5, but not E. coli 026:B6. However, in THP-1 cells, isolated human primary leukocytes, and whole human blood, Ac-hE18A-NH2 reduced responses more strongly than 4F to both E. coli serotypes either when peptides were pre-incubated or co-incubated with LPS, indicating that Ac-hE18A-NH2 also has strong anti-inflammatory effects independent of endotoxin-neutralizing properties. In conclusion, Ac-hE18A-NH2 is more effective than 4F in inhibiting LPS-mediated inflammation, which opens prospective clinical applications for Ac-hE18A-NH2.