Loss of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) in cardiomyocytes results in energy shortage and heart failure. We aim to understand the intracellular signal pathway and extracellular factors regulating Drp1 phosphorylation and mitochondrial morphology and function in cardiomyocytes. We found cyclic mechanical stretching induced mitochondrial fission through Drp1 and focal adhesion kinase (FAK) in neonatal rat ventricular myocytes (NRVMs). FAK regulated phosphorylation of Drp1 and mitochondrial Drp1 levels. Extracellular fibronectin activated Drp1 and caused mitochondrial fission through FAK and extracellular signal-regulated kinase 1/2 (ERK1/2). Fibronectin increased NRVMs oxygen consumption rate and ATP content via FAK-ERK1/2-Drp1. Inhibition of the FAK-ERK1/2-Drp1 pathway caused cellular energy shortage. In addition, the FAK-ERK1/2-Drp1 pathway was rapidly activated by adrenergic agonists and contributed to agonists-stimulated NRVMs respiration. Interestingly, fibronectin limited the adrenergic agonists-induced NRVMs respiration by restricting phosphorylation of Drp1. Our results suggest that extracellular fibronectin and adrenergic stimulations use the FAK-ERK1/2-Drp1 pathway to regulate mitochondrial morphology and function in cardiomyocytes.