Stress is known to cause corpus luteum (CL) dysfunction, and stress hormones play a critical role in this process. However, the mechanism remains unclear. In this study, weaned sows were injected with synthetic adrenocorticotropic hormone (ACTH) for 7 d; wholeâgenome bisulfite sequencing (WGBS) and RNA sequencing was used respectively to investigate the systematic association between ACTH administration and DNA methylation in CL and its relationship to gene expression. Results showed that ACTH treatment significantly increased the concentrations of Cortisol (P < 0.05). The genomeâwide DNA methylation maps of CL were provided, and the global analysis showed the difference between the 2 groups exists in the chromosomes and feature regions of the genome. A total of 88,559 DMRs were identified and the most DMRârelated genes were gathered in terms of metabolic biologic processes, and some DMRârelated genes were involved in cellular differentiation. Nine differentially expressed genes were screened out of coexpressed genes and 4 DMRâassociated genes that were also differentially expressed (P < 0.05). In summary, our study firstly provides insight into the regulation of ACTH administration on genomic DNA methylation and gene expression in CL. We revealed a remarkable alteration of DNA methylation in CL caused by ACTH treatment, and identified 4 DMRârelated genes that may be involved in the CL function under stress conditions.âZhao, F., Wu, W., Wei, Q., Shen, M., Li, B., Jiang, Y., Liu, K., Liu, H. Exogenous adrenocorticotropic hormone affects genomeâwide DNA methylation and transcriptome of corpus luteum in sows. FASEB J. 33, 3264â3278 (2019). http://www.fasebj.org