Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCδ4 are predominantly expressed in testis. Here we report that male mice in which the PLCδ4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCδ4^−/− sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCδ4^−/− sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCδ4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.