Background and purpose: Infertility is a critical public health issue, and half of its causes are related to male factors. Teratozoospermia is one of major cause of male infertility. In our previous studies, we identified the TBC1 domain family member 21 (TBC1D21) gene, a Rab GTPase-activating protein (GAP), as a sterility-related gene in cDNA microarray compared to normal and spermatogenic testicular tissues. TBC1D21 is specifically expressed in post-meiotic male germ cells. Furthermore, the loss of Tbc1d21 allele in mice results in severe defects in the midpiece of the sperm’s tail. Additionally, TBC1D21 interactors were selected using proteomic assays. Herein, we focused on RAB5C, which is a TBC1D21 interactor. Methods and Results: RAB5C was localized in the acrosomal region and elongating tail in spermatids of murine testicular sections. In a mature sperm cell, RAB5C signals were concentrated in the acrosome and were scattered around the midpiece. Interestingly, RAB5C pro-tein levels decreased in the acrosome after triggering acrosomal reaction. Finally, we confirmed that the interspersed RAB5C signals were co-localized with TBC1D21 within the midpieces of the mature sperm cells. Conclusion: Based on these results, we suggest RAB5C, a TBC1D21 interactor, was involved in acrosomal and midpiece formation.