Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-κB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-α and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-κB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-κB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IκB kinase α/β complex and the expression of some NF-κB family members. Treatment of macrophages with the γ-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-κB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-α and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-κB signaling. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.