Fine particulate matter (PM_2.5) exposure is associated with cardiovascular disease (CVD) morbidity and mortality. Mitochondria are sensitive targets of PM_2.5, and mitochondrial dysfunction is closely related to the occurrence of CVD. The epigenetic mechanism of PM_2.5-triggered mitochondrial injury of cardiomyocytes is unclear. This study focused on the miR-421/SIRT3 signaling pathway to investigate the regulatory mechanism in cardiac mitochondrial dynamics imbalance in rat H9c2 cells induced by PM_2.5. Results illustrated that PM_2.5 impaired mitochondrial function and caused dynamics homeostasis imbalance. Besides, PM_2.5 up-regulated miR-421 and down-regulated SIRT3 gene expression, along with decreasing p-FOXO3a (SIRT3 downstream target gene) and p-Parkin expression and triggering abnormal expression of fusion gene OPA1 and fission gene Drp1. Further, miR-421 inhibitor (miR-421i) and resveratrol significantly elevated the SIRT3 levels in H9c2 cells after PM_2.5 exposure and mediated the expression of SOD2, OPA1 and Drp1, restoring the mitochondrial morphology and function. It suggests that miR-421/SIRT3 pathway plays an epigenetic regulatory role in mitochondrial damage induced by PM_2.5 and that miR-421i and resveratrol exert protective effects against PM_2.5-incurred cardiotoxicity.