Spermatogenesis is an elaborately regulated system dedicated to the continuous production of spermatozoa via the genesis of spermatogonia. In this process, a variety of genes are expressed that are relevant to the differentiation of germ cells at each stage. Although Notch signaling pathway plays a critical role in germ cell development in Drosophila and Caenorhabditis elegans, its function and importance for spermatogenesis in mammals is controversial. We report that NKAPL (NFkB Activating Protein-Like) is a novel germ cell-specific transcriptional suppressor in Notch signaling pathway. This protein is presumably originated from NKAP (NFkB Activating Protein) due to its high homology in amino acids and conserved domains. In mice NKAPL is expressed robustly in the testis from age of 3 weeks and localized to the nuclei of spermatogonia and early spermatocytes. We have shown that NKAPL can interact with several molecules of the Notch corepressor complex, such as CIR, HDAC3, and CSL, which regulate the transcription level of the downstream genes. Furthermore, NKAPL suppressed the transcription of Notch signaling pathway through the CSL binding site, a Notch signaling specific binding site, in luciferase assay. In order to understand the function of NKAPL, we have generated Nkapl knockout mice. Male germ cells showed a complete developmental arrest at meiosis, and significant apoptosis was observed at pachytene spermatocytes. Real-time PCR revealed the significant increases of downstream genes in Notch signaling in the testes of the knockout mice, compared to wild type. Additionally, significant changes were found in differentiation markers and apoptotic factors. In summary, our data suggests that NKAPL is indispensable for male fertility, and aberrantly elevated Notch signaling has negative effects on spermatogenesis. Our results add a new dimension to the complexity of Notch signalling within the testis.