Dynamitin (Dmn) is a major component of dynactin, a multiprotein complex playing important roles in a variety of intracellular motile events. We previously found that Wolbachia bacterial infection resulted in a reduction of Dmn protein. As Wolbachia may modify sperm in male hosts, we speculate that Dmn may have a function in male fertility. Here we used nosGal4 to drive Dmn knock down in testes of Drosophila melanogaster to investigate the functions of Dmn in spermatogenesis. We found that knockdown of Dmn in testes dramatically decreased male fertility, overexpression of Dmn in Wolbachia-infected males significantly rescued male fertility, indicating an important role of Dmn in inducing male fertility defects following Wolbachia infection. Some scattered immature sperm with late canoe-shaped head distributed in the end of Dmn knockdown testis and only about half mature sperm were observed in the Dmn knockdown testis relative to those in the control. Transmission electron microscopy (TEM) exhibited fused spermatids in cysts and abnormal mitochondrial derivatives. Immunofluorescence staining showed significantly less abundance of tubulin around the nucleus of spermatid and scattered F-actin cones to different extents in the individualization complex (IC) during spermiogenesis in Dmn knockdown testes, which may disrupt the nuclear condensation and sperm individualization. Since dynein-dynactin complex has been shown to mediate transport of many cellular components, including mRNAs and organelles, these results suggest that Dmn may play an important role in Drosophila spermiogenesis by affecting transport of many important cytoplasmic materials.