The acrosome is a cap-shaped, exocytotic vesicle that is located at the anterior part of the sperm nucleus and plays an essential role in fertilization. An acrosomal protein, ACRBP/sp32, has been initially identified as a binding partner for the precursor (proACR/proacrosin) and intermediate forms of sperm serine protease ACR/acrosin. We previously suggested that ACRBP may participate in the packaging of proACR into the acrosome and may promote autoactivation of proACR during the acrosome reaction. In this study, we produced mice lacking ACRBP to uncover the physiological role of ACRBP in spermatogenesis and fertilization. The ACRBP-deficient mice showed male subfertility. The loss of ACRBP resulted in inadequate condensation and incomplete spread of acrosomal components in the acrosomal granule of round spermatids, leading to the fragmentation of acrosome on the sperm head. The ACRBP-deficient sperm also exhibited defective motility and significantly impaired ability to fertilize the oocytes in vitro. Intriguingly, proACR was already processed and converted into a mature form of ACR in the mutant sperm. To examine whether the observed phenotype of ACRBP-deficient mice is rescued by transgenic introduction of Acrbp into the knockout mice, we generated two transgenic mouse lines expressing wild-type (W) and alternative splice form (V5) of Acrbp. The reduced fertility of ACRBP-deficient males was greatly recovered by the introduction of Acrbp-W and Acrbp-V5. The deformed acrosome of ACRBP-deficient sperm was normally restructured by transgenic expression of Acrbp-V5. Moreover, proACR remained unprocessed in ACRBP-deficient sperm expressing Acrbp-W, similar to wild-type sperm. These results demonstrate that ACRBP-V5 plays a key role in condensation and subsequent spread of acrosomal proteins during spermiogenesis. It is also suggested that ACRBP-W functions in the retention of proACR in the acrosome until sperm undergo acrosomal exocytosis.