Macrophage activation participates pivotally in the pathophysiology of chronic inflammatory diseases including atherosclerosis. Prostaglandin E2 (PGE2) exerts an anti-inflammatory action through EP4 receptor in macrophages. We recently identified a novel EP4 receptor-associated protein, EPRAP, whose function in PGE2- mediated anti-inflammation remains undefined. Here we demonstrate that PGE2 pretreatment selectively inhibits lipopolysaccharide (LPS)-induced nuclear factor kappaB1 (NF-kappaB1) p105 phosphorylation and degradation in mouse bone marrow-derived macrophages (BMDM) through EP4-dependent mechanisms. Similarly, directed EPRAP expression in RAW264.7 cells suppresses LPS-induced p105 phosphorylation and degradation, and subsequent activation of mitogen-activated protein kinase (MAPK)- kinase (MEK) 1/2. Forced expression of EPRAP also inhibits NF-kappaB activation induced by various pro-inflammatory stimuli in a concentration-dependent manner. In cotransfected cells, EPRAP, which contains multiple ankyrin-repeat motifs, directly interacts with NF-kappaB1 p105/p50 and forms a complex with EP4. In EP4-overexpressing cells, PGE2 enhances the protective action of EPRAP against stimulus-induced p105 phosphorylation, whereas EPRAP silencing in RAW264.7 cells impairs the inhibitory effect of PGE2-EP4 signaling on LPS-induced p105 phosphorylation. Additionally, EPRAP knockdown as well as deficiency of NF-kappaB1 in macrophages attenuate the inhibitory effect of PGE2 on LPS-induced MIP-1beta production. Taken together, PGE2-EP4 signaling positively regulates NF-kappaB1 p105 protein stability through EPRAP after pro-inflammatory stimulation, limiting macrophage activation.