Zona pellucida (ZP)–induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCδ4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca 2+ responses in single sperm, and we report that the [Ca 2+ ] i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCδ4 −/− sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca 2+ ] i increases in PLCδ4 −/− sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca 2+ ] i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca 2+ ] i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca 2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCδ4 −/− sperm. These results indicate that PLCδ4 is an important enzyme for intracellular [Ca 2+ ] i mobilization in the ZP-induced acrosome reaction and for sustained [Ca 2+ ] i increases through SOC induced by ZP and progesterone in sperm.