Stresses that perturb the folding of nascent endoplasmic reticulum (ER) proteins activate the ER stress response. Upon ER stress, ER-associated ATF6 is cleaved; the resulting active cytosolic fragment of ATF6 translocates to the nucleus, binds to ER stress response elements (ERSEs), and induces genes, including the ER-targeted chaperone, GRP78. Recent studies showed that nutrient and oxygen starvation during tissue ischemia induce certain ER stress response genes, including GRP78; however, the role of ATF6 in mediating this induction has not been examined. In the current study, simulating ischemia (sI) in a primary cardiac myocyte model system caused a reduction in the level of ER-associated ATF6 with a coordinate increase of ATF6 in nuclear fractions. An ERSE in the GRP78 gene not previously shown to be required for induction by other ER stresses was found to bind ATF6 and to be critical for maximal ischemia-mediated GRP78 promoter induction. Activation of ATF6 and the GRP78 promoter, as well as grp78 mRNA accumulation during sI, were reversed upon simulated reperfusion (sI/R). Moreover, dominant-negative ATF6, or ATF6-targeted miRNA blocked sI-mediated grp78 induction, and the latter increased cardiac myocyte death upon simulated reperfusion, demonstrating critical roles for endogenous ATF6 in ischemia-mediated ER stress activation and cell survival. This is the first study to show that ATF6 is activated by ischemia but inactivated upon reperfusion, suggesting that it may play a role in the induction of ER stress response genes during ischemia that could have a preconditioning effect on cell survival during reperfusion.