AbstractRecycling andde-novodeposition of histones during DNA replication is a critical challenge faced by eukaryotic cells and is coordinated by histone chaperones. Spermatogenesis is highly regulated sophisticated process necessitating not only histone modification but loading of testis specific histone variants. Here, we show that Germ Cell Nuclear Acidic protein (GCNA), a germ cell specific protein in adult mice, can bind histones and purified GCNA exhibits histone chaperone activity. GCNA associates with the DNA replication machinery and supports progression through S-phase in murine undifferentiated spermatogonia (USGs). Whilst GCNA is dispensable for embryonic germ cell development, it is required for the maintenance of the USG pool and for long-term production of sperm. Our work describes the role of a germ cell specific histone chaperone in USGs maintenance in mice. These findings provide a mechanistic basis for the male infertility observed in patients carryingGCNAmutations.