Cholesterol efflux is the key step of reverse cholesterol transport and has become a therapeutic target against atherosclerosis. Human apolipoprotein A-I (apoA-I) is the main protein in high-density lipoprotein (HDL) and has 243 amino acids. ApoA-I mimetic peptides have no sequence homology to apoA-I but possess the class-A amphipathic helical motif that presents in apoA-I lipid binding domains. D-4F is one of the apoA-I mimetic peptides and exerts diverse atheroprotective functions similar to apoA-I. However, the exact role of D-4F on lipid metabolism in macrophages is not clear yet. Therefore, we studied the effect of D-4F on cholesterol efflux, cAMP levels and expression of ATP-binding cassette transporter A1 (ABCA1) in RAW264.7 mouse macrophages. Cells were incubated with 1, 10, 50 or 100 microg/ml D-4F for 24 hours, and the cholesterol efflux was assessed. Here, D-4F significantly increased the cholesterol efflux in concentration- and time-dependent manners. Concomitantly, D-4F increased intracellular cAMP levels and the expression levels of ABCA1 mRNA and protein in a dose-dependent manner, consistent with the increase in the cholesterol efflux from macrophages. 8-Br-cAMP (cAMP activator) increased the D-4F-mediated cholesterol efflux by 39%. Moreover, the increases in cholesterol efflux and ABCA1 expression induced by 8-Br-cAMP could be inhibited by the treatment with H89, a protein kinase A (PKA) inhibitor. In conclusion, these results suggest that the synthetic peptide D-4F promotes cholesterol efflux in macrophages through the cAMP-PKA-ABCA1 pathway, which may open new avenues for the treatment of atherosclerosis.