Objective：This study aims to investigate the role of N6-methyladenosine（m6A）reader protein Ythdf3 during spermatogenesis in knockout mouse model. Methods：We generated Ythdf3 knockout mice by CRISPR/Cas9 technology，and detected the role of Ythdf3 in spermatogenesis by immunofluorescence，HE staining. Results：Immunohistochemistry results showed that Ythdf3 expressed in the nuclear of spermatogonia and spermatocytes；Ythdf3 knockout mice were constructed successfully；HE staining results revealed spermatogenic wave and the morphology of epididymis cauda in Ythdf3 knockout mice were normal compared with control. Conclusion：Depletion of Ythdf3 did not affect mouse spermatogenesis in normal physiological condition.