Macrophage-specific Abca1 (ATP-binding cassette transporter A1) knockout (Abca1-M/-M) mice were generated to determine the role of macrophage ABCA1 expression on plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations of chow-fed Abca1-M/-M and wild type (WT) mice were indistinguishable. Compared to WT, Abca1-M/-M macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apolipoprotein A-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts, without induction of the endoplasmic reticulum (ER) stress. Lipopolysaccharide (LPS)-treated Abca1-M/-M macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the nuclear factor-kB (NF-kB) and mitogen-activated protein kinase (MAPK) pathways, which could be diminished by silencing myeloid differentiation primary-response protein 88 (MyD88) or by chemical inhibition of NF-kB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1-M/-M compared to WT mice. Further, cholesterol depletion of macrophages with methyl-b-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cellular membrane FC and lipid raft content.