Objective：Construct YTH N6-methyladenosine RNA binding protein 1（Ythdf1） knockout mice and study its role during mouse spermatogenesis. Methods：Ythdf1 knockout mice were generated by microinjection of CRISPR/Cas9 into zygote successfully. The role of Ythdf1 in mouse spermatogenesis was detected by immunofluorescence，HE and fertility test. Results：The results of RT-PCR showed that Ythdf1 was expressed in the testis of 1 to 8-week-old male mice. Ythdf1 knockout mice were successfully generated. These knockout mice showed normal spermatogenesis，epididymis morphology and fertility. Conclusion：This study found that deletion of Ythdf1 gene had no effect on spermatogenesis under normal breeding conditions.