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Sertoli cells are essential for testis development and normal spermatogenesis by providing support and nutrients. Pre‐messenger RNA (pre‐mRNA) processing is the basic mechanism required for gene expression, and members of the serine/arginine‐rich protein (SR) family are key components of the machines that perform these basic processing events. Serine/arginine‐rich splicing factor 2 (SRSF2) is an important member of the SR family; however, the physiological functions of SRSF2 in Sertoli cells are still unclear. Here, we found that SRSF2 was localized in the nuclei of Sertoli and germ cells in male mice at all stages by breeding Amh‐Cre mice obtained with Srsf2‐specific knockout in Sertoli cells to define the function of SRSF2 in Sertoli cells. The experimental results showed that specific deletion of SRSF2 impaired fetal Sertoli cell proliferation and induced abnormal apoptosis and severe DNA damage in seminiferous tubules, resulting in severe testicular dysplasia, seminiferous tubule atrophy, and almost no normal seminiferous tubules at postnatal day 14. Eventually, these changes resulted in failure to produce normal sperm and infertility. Further RNA‐seq results showed that many key genes related to proliferation and apoptosis were downregulated; Racgap1 mRNA undergoes exon skipping. Thus, SRSF2‐dependent Sertoli cells are essential for testicular development and male reproduction. [ABSTRACT FROM AUTHOR]