Accumulation of unfolded proteins within the endoplasmic reticulum (ER) activates the unfolded protein response, also known as the ER stress response. We previously demonstrated that ER stress induces transcription of the ER Golgi intermediate compartment protein ERGIC-53. To investigate the molecular events that regulate unfolded protein response-mediated induction of the gene, we have analyzed the transcriptional regulation of ERGIC-53. We found that the ERGIC-53 promoter contains a single cis-acting element that mediates induction of the gene by thapsigargin and other ER stress-causing agents. This ER stress response element proved to retain a novel structure and to be highly conserved in mammalian ERGIC-53 genes. The ER stress response element identified contains a 5'-end CCAAT sequence that constitutively binds NFY/CBF and, 9 nucleotides away, a 3'-end region (5'-CCCTGTTGGCCATC-3') that is equally important for ER stress-mediated induction of the gene. This sequence is the binding site for endogenous YY1 at the 5'-CCCTGTTGG-3' part and for undefined factors at the CCATC 3'-end. ATF6 alpha-YY1, but not XBP1, interacted with the ERGIC-53 regulatory region and activated ERGIC-53 ER stress response element-dependent transcription. A molecular model for the transcriptional regulation of the ERGIC-53 gene is proposed.