The trichothecene mycotoxin deoxynivalenol (DON) induces systemic expression of the interleukin-6 (IL-6) and other proinflammatory cytokines in the mouse. The purpose of this study was to test the hypothesis that DON triggers an endoplasmic reticulum (ER) stress response in murine macrophages capable of driving IL-6 gene expression. DON at concentrations up 5000 ng/ml. was not cytotoxic to peritoneal cells. However, DON markedly decreased protein levels but not the mRNA levels of glucose-regulated protein (GRP) 78 (BiP), a chaperone known to mediate ER stress. Inhibitor studies suggested that DON-induced GRP78 degradation was cathepsin and calpain dependent but was proteosome-independent. RNAi-mediated knockdown of GRP78 resulted in increased IL-6 gene expression indicating a potential downregulatory role for this chaperone. GRP78 is critical to the regulation of the two transcription factors, X-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), which bind to cAMP-response element (CRE) and drive expression of CRE-dependent genes such as IL-6. DON exposure was found to increase IRE1α protein, its modified products spliced XBP1 mRNA and XBP1 protein as well as ATF6. Knockdown of ATF6 but not XBP1 partially inhibited DON-induced IL-6 expression in the macrophages. Three other trichothecenes (satratoxin G, roridin, T-2 toxin) and the ribosome inhibitory protein ricin were also found to induce GRP78 degradation suggesting that other translation inhibitors might evoke ER stress. Taken together, these data suggest that in the macrophage DON induces GRP78 degradation and evokes an ER stress response that could contribute, in part, to DON-induced IL-6 gene expression.