ABCA1 is a key mediator of cholesterol efflux to apoA-I in cholesterol loaded macrophages, a first step of RCT in vivo. Unsaturated fatty acids can inhibit cholesterol efflux from macrophages by increasing degradation of ABCA1. However, the detailed mechanisms of ABCA1 regulation by unsaturated fatty acids are not fully understood. In the present study, we investigated the effects of EPA on ABCA1 expression and ABCA1-dependent cholesterol efflux and examined the role of cAMP/PKA pathway on the regulation of ABCA1 by EPA in THP-1 macrophage-derived foam cells. Results showed that EPA significantly destabilized ABCA1 protein and reduced ABCA1-dependent cholesterol efflux but had no effect on ABCA1 mRNA expression. We also revealed that EPA markedly reduced cAMP level and PKA activity and ABCA1 serine phosphorylation. PKA-specific activation by PKA agonist markedly compensated the down-regulation of ABCA1 serine phosphorylation and ABCA1-mediated cholesterol efflux by EPA, while, siRNA of PKA leaded to reduce of ABCA1 serine phosphorylation and ABCA1-mediated cholesterol efflux more significantly than EPA. However, EPA-Induced enhancement of degradation rate of ABCA1 protein did not change by treatment with PKA agonist or PKA-siRNA. These results provide evidence that EPA may have dual negative effects on ABCA1 activity by decreasing ABCA1 protein level and by reducing PKA-mediated ABCA1 serine phosphorylation in THP-1 macrophage-derived foam cells.