OBJECTIVE: To identify the major causative gene(s) of genuine empty follicle syndrome (GEFS) characterized by oocyte degeneration. DESIGN: Genetic and functional studies. SETTING: University-based reproductive medicine center. PATIENT(S): Thirty-five unrelated women with GEFS and oocyte degeneration. INTERVENTION(S): Whole-exome sequencing (WES) and targeted Sanger sequencing. MAIN OUTCOME MEASURE(S): Variants predicted by software and the functional effects of variants assessed via Western blot and immunofluorescence in Chinese hamster ovary (CHO) cells. RESULT(S): We identified zona pellucida (ZP) gene variants in 18 individuals, which included 20 variants in the ZP1 gene, two variants in the ZP2 gene, and one previously reported recurrent variant in the ZP3 gene. The women carrying ZP variants constituted 51.43% of the GEFS cohort. The ZP1 variants were inherited in an autosomal recessive pattern; the ZP2 and ZP3 variants were inherited in an autosomal dominant pattern. All variants were predicted to be deleterious. Studies in CHO cells suggested that most ZP1 variants led to increased intracytoplasmic protein and some variants influenced the intracellular transportation of other ZP proteins. Variant p.R642Q of ZP2 caused the secretion of ZP2 protein with an increased molecular weight, suggesting altered protein modification. Variant p.I619N of ZP2 resulted in increased ZP2 protein in cell lysate and decreased ZP2 protein in culture medium. These results showed that ZP variants might block the intracellular transportation and secretion of ZP proteins and disrupt the zona pellucida. CONCLUSION(S): We identified novel variants of ZP genes in more than half the cohort with GEFS and oocyte degeneration. Variants of ZP genes caused protein intracellular sequestration and failure to assemble the ZP filaments, resulting in EFS and female infertility. Our findings not only reveal the critical roles of ZP genes but also pave the way for the efficient genetic diagnosis of females with GEFS and oocyte degeneration.