The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways, including activation of nuclear factor κB (NF-κB) signaling, which lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn’s disease and Blau syndrome. We used a genome-wide small interfering RNA screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn’s disease risk were identified in the screen. A comparison of candidates from this screen with other “omics” data sets revealed interconnected networks of genes implicated in NF-κB signaling, thus supporting a role for NOD2 and NF-κB pathways in the pathogenesis of Crohn’s disease. Many of these regulators were validated in secondary assays, such as measurement of interleukin-8 secretion, which is partially dependent on NF-κB. Knockdown of putative regulators in human embryonic kidney 293 cells followed by stimulation with tumor necrosis factor–α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB–mediated inflammation.