Metazoans express three unfolded protein response transducers (IRE1, PERK, and ATF6) ubiquitously to cope with endoplasmic reticulum (ER) stress. ATF6 is an ER membrane-bound transcription factor activated by ER stress-induced proteolysis and has been duplicated in mammals. Here, we generated ATF6α- and ATF6β-knockout mice, which developed normally, and then found that their double knockout caused embryonic lethality. Analysis of mouse embryonic fibroblasts (MEFs) deficient in ATF6α or ATF6β revealed that ATF6α is solely responsible for transcriptional induction of ER chaperones and that ATF6α heterodimerizes with XBP1 for the induction of ER-associated degradation components. ATF6α^−/− MEFs are sensitive to ER stress. Unaltered responses observed in ATF6β^−/− MEFs indicate that ATF6β is not a negative regulator of ATF6α. These results demonstrate that ATF6α functions as a critical regulator of ER quality control proteins in mammalian cells, in marked contrast to worm and fly cells in which IRE1 is responsible.