We show the physiological effects and molecular characterization of overexpression of the catalytic core of mitochondrial DNA (mtDNA) polymerase (pol γ‐α) in muscle of Drosophila melanogaster. Muscle‐specific overexpression of pol γ‐α using the UAS/GAL4 (where UAS is upstream activation sequence) system produced more than 90% of lethality at the end of pupal stage at 25°C, and the survivor adult flies showed a significant reduction in life span. The survivor flies displayed a decreased mtDNA level that is accompanied by a corresponding decrease in the levels of the nucleoid‐binding protein mitochondrial transcription factor A (mtTFA). Furthermore, an increase in apoptosis is detected in larvae and adults overexpressing pol γ‐α. We suggest that the pupal lethality and reduced life span of survivor adult flies are both caused mainly by massive apoptosis of muscle cells induced by mtDNA depletion.