In order to acquire fertilization potential, male germ cells undergo an array of well-orchestrated events to achieve functional competence. Throughout these processes, sperm encounter several cysteine-rich secretory proteins (CRISPs). CRISPs are abundantly expressed in the mammalian male reproductive tract and venom of poisonous reptiles and have ion channel regulatory activity. We have shown that CRISP2 is expressed in the testis and localized within the acrosome, outer dense fibers and the principle piece of the sperm. Loss of CRISP2 function results in male sub-fertility, decreased sperm velocity and a compromised ability for sperm to undergo the acrosome reaction. We identified CRISP2 as a binding partner of the CatSper1 subunit of the sperm-specific CatSper ion channel. This interaction was confirmed by antibody pulldown assay and proximity ligation method. CatSper is a major Ca2+ influx ion channel and has a critical role in regulating sperm motility, thus raising the possibility that CRISP2 plays a critical role in the generation of normal sperm flagella waveform. Commercially available sperm systems do not allow a precise analysis of sperm motility. As such, in order to precisely define the role of CRISP2 in sperm motility, we developed a novel high-speed, high-resolution imaging method. We analyzed the sperm from Crisp2-/-and WT mice and found that sperm from Crisp2-/- mice have a motility pattern referred to as ‘stiff mid-piece syndrome,’ consistent with the proposed role of CRISP2 in regulating Ca2+ influx in sperm. In summary, our data show that CRISP2 is required for optimal sperm function, fertility and that it potentially functions via CatSper to control Ca2+ and thus flagella waveform.