Fertilization involves a series of molecular events immediately following egg-sperm fusion; Ca2+ oscillations are the earliest signaling event and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.