Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is highly expressed in myocardium, but little is known about its function in myocytes. Recently, we reported that MCL-1 is essential for myocardial homeostasis and autophagy. Cardiac-specific deletion of MCL-1 in mice led to rapid mitochondrial dysfunction, hypertrophy, and lethal cardiomyopathy. Despite extensive mitochondrial damage, MCL-1 deficient hearts failed to activate mitochondrial autophagy. Parkin, an E3 ubiquitin ligase, normally translocates to damaged mitochondria to promote mitochondrial autophagy, but loss of MCL-1 resulted in cytosolic accumulation of Parkin. However, we found no evidence that MCL-1 functions as a mitochondrial Parkin receptor or substrate. Instead, loss of MCL-1 reduced mitochondrial accumulation of PINK1, which is involved in Parkin recruitment. Additionally, we identified mitochondrial outer membrane (OM) and matrix isoforms of MCL-1 in mouse hearts and found that the two forms respond differently to ischemic injury. Four hours after myocardial infarction, MCL-1OM levels were reduced by 40% in border zone tissue. After 24 hours, MCL-1OM levels returned to baseline. Meanwhile, MCL-1Matrix levels were preserved at four hours, and increased significantly compared to control 24 hours after infarction. These changes correlated with increased expression of HSP70, a chaperone protein that stabilizes MCL-1 and participates in import of mitochondrial proteins. Overexpression of MCL-1Matrix promoted mitochondrial fusion in fibroblasts under baseline conditions and protected cells against FCCP-mediated mitochondrial fission. While 94.1% (1269 of 1349) of control cells exhibited fragmented mitochondria after two hours of FCCP treatment, mitochondria were only fragmented in 41.3% (391 of 947) of cells overexpressing MCL-1Matrix. These data suggest MCL-1 isoforms play different roles in the cellular stress response. MCL-1OM protects against apoptosis, whereas MCL-1Matrix protects mitochondria by promoting fusion. In addition, upregulation of HSP70 may preserve mitochondrial function and cell viability in damaged cardiac myocytes by increasing the stability and mitochondrial import of MCL-1.