Preventing cyclophilin D (cypD) translocation to the inner mitochondrial membrane can limit lethal reperfusion injury through the inhibition of the opening of the mitochondrial permeability transition pore. Inhibition or loss of function of cypD may also result into an endoplasmic reticulum (ER) stress that has been shown to alter cell survival. We therefore questioned whether ER stress might play a role in the protection induced by CypD deficiency or inhibition. CypD-KO and NIM811 (a CypD inhibitor)-treated mice were subjected to a prolonged ischemia-reperfusion (I/R). Area at risk and infarct size was measured using blue dye and triphenyltetrazolium chloride staining. ER stress markers were measured in the hearts during the reperfusion phase. As expected, cypD-KO mice exhibited a decreased infarct size when compared to wild-type mice (8 ± 1 vs. 20 ± 4% of left ventricular weight; p < 0.01). CypD-deficient mice displayed an increased expression of ER stress proteins such as eukaryotic initiation factor 2α (eIF2α) or glucose regulated protein 78 (Grp78 or Bip). The ER stress inhibitor TUDCA prevented the infarct size reduction afforded by the loss of cypD function (mean infarct size averaged 21 ± 4% of LV weight, p < 0.01 vs. cypD-KO). Similar results were obtained when NIM811, an analog of cyclosporine A, was used to pharmacologically (instead of genetically) inhibit cypD function. This study suggests that the ER stress induced by the inhibition of cypD function plays a key role in protecting the heart against lethal ischemia-reperfusion injury.