Chronic exposure to gut bacteria and bacterial products including Nod2 ligands triggers homeostatic regulation in response to various mucosal insults. Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokines via bacterial pattern recognition. On the assumption that ATF3 can be a critical modulator of epithelial inflammation, chronic stimulation of Nod2 was assessed for its effects on ATF3 and proinflammatory signals in response to mucosal ribotoxic insult, which is a critical etiological factor of human intestinal inflammatory disease. Muramyl dipeptide, the minimal moiety of bacterial peptidoglycan, is the Nod2 ligand, and pre-exposure to it enhanced ATF3 expression in ribotoxic stress-exposed human enterocytes. In terms of gene regulation, Nod2 preactivation potentiated ATF3 induction by enhancing stability of the ATF3 transcript, which was particularly linked to the regulation of the 3'-untranslated region of the human ATF3 gene. Moreover, chronic stimulation of Nod2 enhanced both the basal and the ribotoxic stress-stimulated cytoplasmic translocation of the HuR protein, which bound to and stabilized ATF3 messenger RNA (mRNA). Functionally, chronic stimulation of Nod2 also led to superinduction of proinflammatory chemokine genes by the mucoactive ribotoxic stress. However, the chemokine superinduction was not affected by ATF3 gene regulation although Nod2-triggered ATF3 had suppressive effects on the proinflammatory nuclear factor kappa B (NF-κB) signal. This paradoxical superinduction of chemokines was also mediated by enhanced mRNA stabilization by HuR protein in spite of ATF3-mediated suppression of NF-κB signal in human intestinal epithelial cells.