Apolipoprotein A‐I (ApoA‐I) is the main functional protein component of human high‐density lipoproteins. ApoA‐I shows various anti‐inflammatory and atheroprotective properties toward macrophages; however, endogenous apoA‐I expression has not been investigated in macrophages. We have shown that endogenous apoA‐I gene is expressed in human macrophages at both mRNA and protein levels. Endogenous ApoA‐I is localized in intracellular vesicles and at the external side of the plasma membrane in association with ATP‐binding cassette transporter A1 (ABCA1) and lipid rafts in macrophages. We have shown that endogenous ApoA‐I stabilizes ABCA1, moreover, down‐regulation of ApoA‐I by siRNA results in an increase of Toll‐like receptor 4 (TLR4) mRNA and membrane surface protein expression, as well as an enhancement of bacterial lipopolysaccharide (LPS)‐induced expression of tumor necrosis factor‐α (TNF‐α), interleukin 1β (IL‐1β), and inducible nitric oxide synthase (NOS2) genes in human macrophages. TNF‐α stimulates ApoA‐I expression and secretion (1.2±0.2 vs. 4.3±0.9 ng/mg total protein) in macrophages. Obtained results suggest that endogenous ApoA‐I has anti‐inflammatory properties, presumably due to ABCA1 stabilization in macrophages; these results elucidate the cell type‐specific mechanism of the TNF‐α‐mediated regulation of apoA‐I gene expression in monocytes and macrophages.—Mogilenko, D. A., Orlov, S. V., Trulioff, A. S., Ivanov, A. V., Nagumanov, V. K., Kudriavtsev, I. V., Shavva, V. S., Tanyanskiy, D. A., Perevozchikov, A. P. Endogenous apolipoprotein A‐I stabilizes ATP‐binding cassette transporter A1 and modulates Toll‐like receptor 4 signaling in human macrophages. FASEB J. 26, 2019‐2030 (2012). www.fasebj.org