Germ cell maturation is essential for spermatogenesis and testis homeostasis. ATP synthase serves significant roles in energy storage in germ cell survival and is catalyzed by alterations in the mitochondrial membrane proton concentration. The intrinsic cellular mechanisms governing stem cell maturation remain largely unknown. In the present study, in vivo RNA interference (RNAi) screening of major ATP synthase subunits was performed, and the function of ATP synthase for male fertility and spermatogenesis in Drosophila was explored. A Upstream Activation Sequence/Gal4 transcription factor system was used to knock down gene expression in specific cell types, and immunofluorescence staining was conducted to assess the roles of ATP synthase subunits in Drosophila testes. It was identified that knockdown of ATP synthase resulted in male infertility and abnormal spermatogenesis in Drosophila testes. In addition, knockdown of the ATP synthase β subunit in germ cells resulted in defects in male infertility and germ cell maturation, while the hub and cyst cell populations were maintained. Other major ATP synthase subunits were also examined and similar phenotypes in Drosophila testes were identified. Taken together, the data from the present study revealed that ATP synthase serves important roles for male fertility during spermatogenesis by regulating germ cell maturation in Drosophila testes.