Objective-The apolipoprotein A-I (apoA-I) mimetic peptide 4F favors the differentiation of human monocytes to an anti-inflammatory phenotype and attenuates lipopolysaccharide (LPS)-induced inflammatory responses. We investigated the effects of LPS on the Toll-like receptor (TLR) signaling pathway in 4F-differentiated monocyte-derived macrophages. Methods and Results-Monocyte-derived macrophages were pretreated with 4F or vehicle for 7 days. 4F downregulated cell-surface TLRs (4, 5, and 6) as determined by flow cytometry. 4F attenuated the LPS-dependent upregulation of genes encoding TLR1, 2, and 6 and genes of the MyD88-dependent (CD14, MyD88, TRAF6, interleukin-1 receptor-associated kinase 4, and inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta) and MyD88-independent (interferon regulatory factor 3, TANK-binding kinase 1, and Toll-interleukin 1 receptor domain-containing adaptor-inducing interferon-β) pathways as determined by microarray analysis and quantitative reverse transcriptase polymerase chain reaction. Functional analyses of monocyte-derived macrophages showed that 4F reduced LPS-dependent TLR4 recycling, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, activation and translocation of nuclear factor-κB and inhibited the secretion of tumor necrosis factor-α and interleukin-6 induced by LPS or lipoteichoic acid. These changes were associated with depletion of cellular cholesterol and caveolin, components of membrane lipid rafts. Conclusion-These data suggest that disruption of rafts by 4F alters the assembly of TLR-ligand complexes in cell membranes and inhibits proinflammatory gene expression in monocyte-derived macrophages, thus attenuating the responsiveness of macrophages to LPS. © 2012 American Heart Association, Inc.