Objective: To explore the association of circadian rhythm disruption with polycystic ovary syndrome (PCOS) and the potential underlying mechanism in ovarian granulosa cells (GCs). Design: Multicenter questionnaire-based survey, in vivo and ex vivo studies. Setting: Twelve hospitals in China, animal research center, and research laboratory of a women's hospital. Patients/Animals: A total of 436 PCOS case subjects and 715 control subjects were recruited for the survey. In vivo and ex vivo studies were conducted in PCOS-model rats and on ovarian GCs collected from women with PCOS and control subjects. Intervention(s): The PCOS rat model was established with the use of testosterone propionate. Main Outcome Measure(s): Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), RNA sequencing, rhythmicity analysis, functional enrichment analysis. Result(s): There was a significant correlation between night shift work and PCOS. PCOS-model rats presented distinct differences in the circadian variation of corticotropin-releasing hormone, adrenocorticotropic hormone, prolactin, and a 4-h phase delay in thyrotropic hormone levels. The motif enrichment analysis of ATAC-seq revealed the absence of clock-related transcription factors in specific peaks of PCOS group, and RNA sequencing ex vivo at various time points over 24 hours demonstrated the differential rhythmic expression patterns of women with PCOS. Kyoto Encyclopedia of Genes and Genomes analysis further highlighted metabolic dysfunction, including both carbohydrate and amino acid metabolism and the tricarboxylic acid cycle. Conclusion(s): There is a significant association of night shift work with PCOS, and genome-wide chronodisruption exists in ovarian GCs. © 2020 American Society for Reproductive Medicine