Emerging evidence suggest that the abnormal mitochondrial fission participates in pathogenesis of cardiac diseases, including myocardial infarction and heart failure. However, the molecular components regulating mitochondrial network in heart remain largely unidentified. Here we report that NFAT4, miR-324-5p and mitochondrial fission regulator 1 (Mtfr1) function in one signaling axis that regulates mitochondrial morphology and cardiomyocyte cell death. Knocking down Mtfr1 suppresses mitochondrial fission, apoptosis and myocardial infarction. Mtfr1 is a direct target of miR-324-5p, and miR-324-5p attenuates mitochondrial fission, cardiomyocyte apoptosis and myocardial infarction by suppressing Mtfr1 translation. Finally, we show that transcription factor NFAT4 inhibits miR-324-5p expression. Knockdown of NFAT4 suppresses mitochondrial fission and protects cardiomyocyte from apoptosis and myocardial infarction. Our study defines the NFAT4/ miR-324-5p/Mtfr1 axis, which participates in the regulation of mitochondrial fission and cardiomyocyte apoptosis, and suggests potential new treatment avenues for cardiac diseases.