Objective A sperm-specific phospholipase C zeta-1 (Plcz1^{- / -} ) gene knockout mouse was generated by the CRISPR/ Cas9 system to explore the role of the Plcz1 gene in male mouse fertility. Methods Exons 6 and 7 of the mouse Plcz1 gene were selected as target sites to design two pairs of sgRNAs. The two pairs of sgRNAs were inserted into the pX330A plasmid by the Golden Gate method to construct the pX330-sgRNA recombinant plasmid. After amplification and purification, the recombinant plasmid was microinjected into pronuclear of mouse zygote. The embryos were transferred to surrogate mother mice. After the birth of F0 generation mice, tail DNA was analyzed by PCR and sequencing. The F0 generation was mated with wild-type mice to obtain F1 generation mice. The F1 generation was bred to the F2 generation to obtain a homozygous strain of Plcz1 gene knockout mice. To analyze Plcz1^{- / -} male mouse fertility, Plcz1^{- / -} mice were mated with wild-type mice. Results The recombinant plasmid pX330-sgRNA was constructed successfully, and three Plcz1^{- / -} male mice (Plcz1^m3 , Plcz1^m4 and Plcz1^m5 ) were obtained by breeding. Target gene sequencing of the three mice showed that Plcz1^m3 had a 3078 nucleotide deletion between exons 6 and 7 of Plcz1, Plcz1^m4 had a seven nucleotide deletion in exon 7 of Plcz1, which died during feeding, and Plcz1^m5 had a one nucleotide deletion in exon 6 of Plcz1. Conclusions Plcz1^{- / -} male mice were successfully generated using the CRISPR/ Cas9 system. Plcz1^{- / -} male mice were fertile, but their fertility was significantly decreased compared with wild-type mice.