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Broodiness in egg-laying hens (EHs) leads to ovarian atrophy, resulting in reduced egg-laying performance. However, the ovarian regulatory mechanisms in broody hens (BCs) remain elusive. Therefore, ovaries were removed from 300-day-old BCs and EHs for RNA sequencing. Ovarian morphology and histological characteristics of the BC and EH groups were compared and analyzed. The EH group had significantly more hierarchical follicles (HFs) and small yellow follicles (SYFs) than that of the BC group. Although several secondary follicles (SFs) and primary follicles were observed in the ovaries of the EH group, only a few SFs were observed in the ovaries of the BC group. Subsequently, RNA-sequencing analysis was conducted to determine the ovarian expression profiles of the two groups. Transcriptome sequencing identified 259 differentially expressed genes (DEGs) between the BC and EH groups. Of the 259 DEGs, 136 were upregulated and 123 were downregulated. The DEGs were mapped to 22 gene ontology terms and 4 Kyoto Encyclopedia of Genes and Genomes pathways for ovarian tissue. The analysis showed that matrix metalloproteinases 11/13 (MMP11/MMP13) were enriched in the extracellular matrix. The extracellular matrix mediated by MMP13 is affected by follicle-stimulating hormone, prolactin, and estrogen, which are critical signaling pathways that may affect ovarian follicle development to regulate the large yellow follicle reserve process and the ovulation cycle of broody Chahua chickens. These findings indicate that understanding differences in gene expression between the ovarian tissues of BCs and EHs could serve as a valuable reference point for enhancing egg-laying performance in Chahua chickens., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest., (2024 Japan Poultry Science Association.)