The regulatory elements controlling gene expression during acute inflammation are not fully elucidated. Here we report the identification of a set of NF-κB-bound elements and common chromatin landscapes underlying the acute inflammatory response across cell-types and mammalian species. Using primary vascular endothelial cells (human/mouse/bovine) treated with the pro-inflammatory cytokine, Tumor Necrosis Factor-α, we identify extensive (~30%) conserved orthologous binding of NF-κB to accessible, as well as nucleosome-occluded chromatin. Regions with the highest NF-κB occupancy pre-stimulation show dramatic increases in NF-κB binding and chromatin accessibility post-stimulation. These 'pre-bound' regions are typically conserved (~56%), contain multiple NF-κB motifs, are utilized by diverse cell types, and overlap rare non-coding mutations and common genetic variation associated with both inflammatory and cardiovascular phenotypes. Genetic ablation of conserved, 'pre-bound' NF-κB regions within the super-enhancer associated with the chemokine-encoding CCL2 gene and elsewhere supports the functional relevance of these elements.