Myocardial infarction is a leading cause of mortality worldwide. Here we report that modulation of microRNA-499 (miR-499) levels affects apoptosis and the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. We found that both the [alpha]- and [beta]-isoforms of the calcineurin catalytic subunit are direct targets of miR-499 and that miR-499 inhibits cardiomyocyte apoptosis through its suppression of calcineurin-mediated dephosphorylation of dynamin-related protein-1 (Drp1), thereby decreasing Drp1 accumulation in mitochondria and Drp1-mediated activation of the mitochondrial fission program. We also found that p53 transcriptionally downregulates miR-499 expression. Our data reveal a role for miR-499 in regulating the mitochondrial fission machinery and we suggest that modulation of miR-499 levels may provide a therapeutic approach for treating myocardial infarction.