Ribotoxic stress responses lead to the expression of genes important for cellular homeostasis by modulating cell survival, proliferation and differentiation. ATF3 was investigated for its modulation of the epithelial cellular integrity in response to mucosal ribotoxic stress. ATF3 expression was up-regulated by chemical agents causing ribotoxic stress such as deoxynivalenol and anisomycin in different types of intestinal epithelial cells. Moreover, reduction of ATF3 gene expression promoted ribotoxic stress-triggered programmed cell death, implicating a protective role of ATF3 in epithelial cell survival. Mechanistically, stabilization of ATF3 messenger RNA and protein played a critical role in maintaining enhanced levels of ATF3 production in response to the ribotoxic chemical agent. For ATF3 mRNA stability, p38 specific inhibitor SB203580 was the most efficient agent for suppression, suggesting the involvement of the p38 MAP kinase in ATF3 mRNA stabilization. In addition, the p38 MAP kinase as well as its downstream mediator glycogen synthase kinase 3beta (GSK3beta) was involved in ATF3 protein stabilization caused by chemical ribotoxic stress in human epithelial cells. As another separate signaling cascade, double-stranded RNA (dsRNA)-activated protein kinase (PKR) was demonstrated to translationally modulate ATF3 expression and contribute to the epithelial cell survival. PKR interference caused cells to be more susceptible to cell death caused by the chemical ribotoxic stress. The results of this study showed that enhanced ATF3 production was associated with cellular defenses by maintaining the epithelial survival after ribotoxic mucosal insults.