Objective: Mitochondrial dysfunction has been identified in various diseases including diabetes and heart diseases. Although mitochondria DNA (mtDNA) encodes 13 minimal proteins involved in oxidative phosphorylation, mutation or depletion of mtDNA could cause severe mitochondrial malfunction originated from mitochondria itself and crosstalk between nucleus and mitochondria. However, it is largely unknown what changes would be happened when mtDNA was depleted. Methods: We established mtDNA depleted rat myoblast-like H9c2 cells by supplementing with ethidium bromide and uridine. After confirming depletion of mtDNA with quantitative PCR and gel electrophoresis, we investigated changes in mitochondrial physical parameters using flowcytometry and resistance to serum starvation and sodium nitroprusside insults. Results: The mtDNA depeletion led to changes in cell morphology. The mtDNA depleted H9c2 cells showed lower in mitochondrial membrane potential, cardiolipin, calcium and free zinc contents than h9c2 naïve cells. Cytosolic and mitochondrial reactive oxygen species was significantly higher in mtDNA depleted H9c2 cells. Although oxygen consumption rate and cell proliferation were decreased, mtDNA depleted H9c2 cells were more resistant against serum deprivation and nitroprusside insults than h9c2 naïve cells. In addition, mtDNA depleted cells showed higher in nuclear encoded acetyl-CoA carboxylase and cyclophilin D protein level. Conclusion: Taken together, mtDNA depletion caused drastic changes in tested mitochondrial physical parameters and these changes may be involved in disturbance in mitochondrial homeostasis and may lead to pathophysiological predisposition.