Perturbation of the endoplasmic reticulum (ER) results in a conserved stress response called the unfolded protein response (UPR). Macrophages undergoing a UPR respond to LPS with log-fold increased production of IFN-beta, a cytokine with diverse roles in innate and adaptive immunity. In this study, we found that thapsigargin-induced ER stress augmented recruitment of IFN regulatory factor-3, CREB binding protein/p300, and transcriptional machinery to the murine ifnb1 promoter during LPS stimulation. Although full synergistic IFN-beta production requires X-box binding protein 1 (XBP-1), this UPR-regulated transcription factor did not appreciably bind the ifnb1 promoter. However, XBP-1 bound a conserved site 6.1 kb downstream of ifnb1, along with IFN regulatory factor-3 and CREB binding protein only during concomitant UPR and LPS stimulation. XBP-1 physically associates with p300, suggesting a mechanism of multimolecular assembly at the +6.1 kb site. Luciferase reporter assays provide evidence this +6 kb region functions as an XBP-1-dependent enhancer of ifnb1 promoter activity. Thus, this study identifies a novel role for a UPR-dependent transcription factor in the regulation of an inflammatory cytokine. Our findings have broader mechanistic implications for the pathogenesis of diseases involving ER stress and type I IFN, including viral infection, ischemia-reperfusion injury, protein misfolding, and inflammatory diseases.