Inter-organelle contact and communication between mitochondria (Mito) and endoplasmic reticulum (ER), also known as sarcoplasmic reticulum (SR) in cardiomyocytes (CM), play vital roles in the maintenance of Mito and SR properties and cellular homeostasis. Here, we tested the hypothesis that the formin, Diaphanous-1 (DIAPH1), which regulates actin dynamics and signal transduction, contributes to these processes. Mice bearing CM-specific deletion of Diaph1 displayed reduced injury and superior functional recovery after the induction of cardiac ischemia and reperfusion (I/R). Studies in DIAPH1-silenced human induced pluripotent stem cell-derived CMs (HiPSC-CMs) and murine hearts devoid of CM-Diaph1 revealed that DIAPH1 interacts directly with Mitofusin-2 (MFN2) to regulate Mito-SR contact, Mito turnover, mitophagy, and oxidative stress. Ischemic murine hearts and human heart biopsies revealed more robust DIAPH1-MFN2 interaction vs. that observed in non-ischemic hearts. Solution structure studies affirm this interaction and reveal strong pH-dependent interaction between the Diaphanous Inhibitory Domain (DID) and the cytosolic GTPase domain of MFN2. In HiPSC-CMs, introduction of synthetic linkers, which reduce the Mito-SR distance, mitigated the cardioprotective benefits of Diaph1 deletion. Finally, DIAPH1 binding to the cytoplasmic domain of receptor for advanced glycation endproduct (RAGE) supports signal transduction and contributes to I/R injury in the heart; in HiPSC-CMs, small molecule antagonism of RAGE-DIAPH1 reduced DIAPH1-MFN2 interaction and increased Mito-SR distance. This work establishes fundamental roles for DIAPH1-MFN2 interaction in the regulation of Mito-SR contact networks that control responses to ischemic stress. Targeting pathways that regulate DIAPH1-MFN2 may facilitate recovery from cardiac ischemic injury. Overall design: Two by two factorial experiment with DIAPH1 knockdown, scramble control, DIAPH1 knockdown under hypoxia, and scramble control under hypoxia. Four biological replicates per experimental group. All samples were pooled for Illumina sequencing in a single lane, and the resulting pool was run twice on different days, creating technical replicates for all samples.