Male infertility is a global public health issue and contributes to nearly half of all infertility cases. Small GTP-binding proteins are essential for numerous cellular processes including spermatogenesis. We have identified that TBC1D21 gene (also named as Male Germ Cell RAB GTPase-activating protein, MGCRABGAP) is a novel germ cell specific GTPase-activating proteins and down-regulated in testicular tissues of the infertile men. Until now, the spermatogenic functions of TBC1D21 are still known. In present study, we demonstrate that TBC1D21 is critical for multiple aspects of sperm tail integrity, such as the arrangement and morphology of mitochondria and the integrity of axoneme microtubules. In the Tbc1d21-/- mice, the sperm number is only slightly lower than in wild-type mice but most of the sperm from Tbc1d21-/- mice display the severe impaired tail structure and shorter mitochondria sheath in the midpiece. Ultrastructure examination revels the disordered arrangement and irregular morphology of mitochondria and disorganized microtubules in sperm flagellum. We also identified the interacting partners of TBC1D21 through co-IP and following by LC-MS/MS. Among these interactors, translocase of outer membrane 22 (TOM22) and dynein heavy chain 7 (DNAH7) are the components of mitochondria and axoneme, respectively. In mature sperm, TBC1D21 interacts with TOM22 and colocalizes with TOM22 and DNAH7 in the region of midpiece. In addition, loss of TBC1D21 cause the dispersed localization of TOM22 and DNAH7 from the midpiece. Based on these results, we suggest that TBC1D21 is a key regulator for maintaining the integration of sperm tail through its interactors.