The dynamic and reversible regulation roles of m6A modification and the characterization of m6A readers have provided new insights into spermatogenesis at the post-transcriptional level.YTHDF2,as an meA reader,has been reported to mediate the meA-containing transcript decay during the mouse oocyte maturation,embryonic stem cell differentiation,neural development,and zebrafish maternal-to-zygotic transition.However,the roles of YTHDF2 in mammalian sper-matogenesis are uncertain.Here,we generated germ cell-specific Ythdf2 mutants (Ythdf2-vKO)at a C57BL/6J background and demonstrated that YTHDF2 is essential for mouse spermatogen-esis and fertility.Ythdf2-vKO provides oligoasthenoteratozoospermia phenotype with increased apoptosis in germ cells.High-throughput RNA-seq analysis showed that a group of mRNAs is upregulated in Ythdf2-vKO mouse testis;further analysis and MeRIP-qPCR data showed that most of the upregulated genes in Ythdf2-vKO mouse testis are modified with m6A and are YTHDF2 candidate binding genes.Interestingly,RNA-seq analysis combined with our previous single-cell transcriptomics data of mouse spermatogenesis pointed out the failure of a wave of transcript transition during the spermatogenesis of Ythdf2-vKO mice,which was confirmed by gene expres-sion analysis using qPCR of diplotene spermatocytes and round spermatids obtained through fluorescence-activated cell sorting.Our study demonstrates the fundamental role of YTHDF2 dur-ing mouse spermatogenesis and provides a potential candidate for the diagnosis of male infertility with the oligoasthenoteratozoospermia syndrome.