Ring1 and Yin Yang 1-Binding Protein (RYBP) is a member of non-canonical polycomb repressive complex 1 to mediate monoubiquitination of histone H2A at lysine 119. It plays an important role in development, but its role in reproduction remains illusive. In this study, we used Rybp conditional knockout mouse model to genetically ablate Rybp in male germ cells. We found that Rybp deficiency during spermatogenesis led to smaller testes, loss of germline cells, disturbed meiosis, increased apoptosis of spermatocytes, decreased sperm motility, and reduced global H3K9me3, without impacting retrotransposon expression. Meanwhile, we depleted Rybp during oogenesis, but oocyte maturation and preimplantation development were normal. Our findings demonstrate that RYBP plays important roles in spermatogenesis through regulating meiosis and sperm motility.