Using conditional knockout mice model, here we report the essential role of DIS3L2 in regulating male germline proliferation, growth and differentiation to ensure male fertility. We document that DIS3L2 is essential for spermatogonial homeostasis. DIS3L2-specific deletion in spermatogonia results in the reduction of the undifferentiated spermatogonial population and the block in transition to the differentiating stage in postnatal testes. Overall design: RNA-seq was performed using postnatal day 4 (P4) testes between control and Dis3l2 cKO mice in order to identify the differentially expressed genes.