During multicellular organism reproduction, organelles, cytoplasmic materials, and mitochondrial DNA (mtDNA) are all derived from maternal lineage. Active mtDNA elimination during spermatogenesis has emerged as a conserved mechanism ensuring the uniparental mitochondrial inheritance in animals (1-4). However, given the existence of post-fertilization processes degrading sperm mitochondria (5-9), the physiological significance of sperm mtDNA removal is not clear. We report that mtDNA removal is indispensable for sperm development and activity. We uncover a novel mitochondrial exonuclease, ExoA (Exonuclease A) that is specially expressed in late spermatogenesis and exclusively required for mtDNA elimination. Loss of ExoA impairs mtDNA clearance in elongated spermatids and impedes the progression of individualization complexes that strip away cytoplasmic materials and organelles. Additionally, persistent mtDNA in mature sperm causes marked fragmentation of nuclear genome and complete sterility of exoA mutant male flies. All these defects can be suppressed by expressing a mitochondrially targeted bacterial exonuclease to ectopically remove mtDNA. Our work illustrates the developmental necessity of mtDNA clearance for the effective cytoplasm removal at the end of spermatid morphogenesis and to prevent potential nuclear-mito imbalance in mature sperms, in which the activity of nuclear genome is shutdown. Hence, the uniparental mitochondrial inheritance seems co-evolved with a key feature of sexual reproduction, the asymmetry between two gametes.