Abstract The zona pellucida (ZP) plays vital roles in reproductive processes including oogenesis, fertilization, and preimplantation development. Both human and rat ZP consist of four glycoproteins, called ZP1, ZP2, ZP3, and ZP4. Our previous research reported a novel Zp1 mutation in cases of human infertility, associated with an abnormal phenotype involving the absence of the ZP. Here, we developed a homologous rat strain to investigate the pathogenic effect. The ovaries of homozygous (Zp1MT/MT) females possessed both growing and fully grown oocytes; the oocytes completely lacked a ZP, but ZP1 was detectable inside the cytoplasm. Only 1–2 eggs were recovered from oviducts of superovulated Zp1MT/MT females, while an average of 21 eggs were recovered from superovulated Zp1WT/WT per female. The eggs of Zp1MT/MT females were not surrounded by a ZP and lost their fertilization capacity in vitro. Zp1MT/MT females mated with wild-type males failed to become pregnant. Studies in 293T cells showed that mutant Zp1 resulted in a truncated ZP1 protein, which might be intracellularly sequestered and interacted with wild-type ZP3 or ZP4. Our results suggest that the Zp1 point mutation led to infertility and loss of the ZP in oocytes in rats.