Continuous mitotic and meiotic cycles are essential prerequisites for germ cell development and male fertility. However, our understanding of the mechanisms underlying spermatogonial differentiation and meiosis in mammals remains limited. In this study, we employed a unique gene knockout mouse model to elucidate the critical role of heterogeneous nuclear ribonucleoprotein K (hnRNPK) in spermatogenesis and male fertility. Specifically, adult mice with germ cell-specific hnRNPK deletion exhibited complete infertility, with nearly a complete absence of spermatocytes in the seminiferous tubules, leaving only a small number of pre-leptotene spermatocytes. Further studies revealed that hnRNPK knockout disrupted spermatogonial differentiation and blocked entry into meiosis. Single-cell RNA sequencing analysis indicated that hnRNPK deletion led to cell cycle abnormalities in differentiating spermatogonia, which were unable to entry to meiosis and instead underwent apoptosis. Additionally, hnRNPK was found to primarily regulate post-transcriptional processes in germ cells. In the cytoplasm, hnRNPK interacts with ribosomal proteins (RPS3A, RPL13) and translation initiation factors (EIF4A1, EIF4A3) to regulate the initiation of translation. Following hnRNPK deletion, the translational efficiency of spermatogenesis-related genes was significantly reduced. In the nucleus, hnRNPK interacts with splicing factors and participates in the splicing of target genes related to differentiation and meiosis. This study highlights the functional role and mechanistic involvement of hnRNPK in spermatogenesis and provides valuable insights into the post-transcriptional regulatory mechanisms that govern this process.