Cellular inhibitor of apoptosis proteins (cIAPs) block apoptosis, but their physiological functions are still under investigation. Here, we report that cIAP1 and cIAP2 are E3 ubiquitin ligases that are required for receptor-interacting protein 2 (RIP2) ubiquitination and for nucleotide-binding and oligomerization (NOD) signaling. Macrophages derived from Birc2−/− or Birc3−/− mice, or colonocytes depleted of cIAP1 or cIAP2 by RNAi, were defective in NOD signaling and displayed sharp attenuation of cytokine and chemokine production. This blunted response was observed in vivo when Birc2−/− and Birc3−/− mice were challenged with NOD agonists. Defects in NOD2 signaling are associated with Crohn's disease, and muramyl dipeptide (MDP) activation of NOD2 signaling protects mice from experimental colitis. Here, we show that administration of MDP protected wild-type but not Ripk2−/− or Birc3−/− mice from colitis, confirming the role of the cIAPs in NOD2 signaling in vivo. This discovery provides therapeutic opportunities in the treatment of NOD-dependent immunologic and inflammatory diseases.