Mitochondria serve as the energy provider and enable life activities, and they are the only organelles containing extra-chromosomal DNA. Each mitochondrion contains multiple copies of its genome, which is usually referred to as mitochondrial DNA (mtDNA). mtDNA encodes necessary electron transport chain complex subunits, as well as the essential RNAs for their translation within the organelle. Therefore, the precondition for intact mitochondrial function and cardiomyocyte survival is the integrity of mtDNA. Accumulating evidence suggests that the disruption of mtDNA integrity is involved in ischemia/reperfusion-induced mitochondrial dysfunction and cardiomyocyte injury. Here, we review the current opinions about the pathways of mtDNA integrity maintenance and discuss the role of mtDNA integrity in cardiomyocyte injury reacting to ischemia/reperfusion. We also discuss the mechanisms by which mtDNA mediates ischemia/reperfusion-induced cardiomyocyte injury, together with therapeutic strategies by targeting mtDNA.