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The neuroendocrine system is key in regulating systemic physiological processes and milk synthesis. However, the hypothalamic\u2010pituitary axis response to HS is still unclear. In this study, a group of lactating mice underwent a daily 2\u2010h heat treatment (36\u00b0) for 14 d to explore possible cross\u2010talk between the hypothalamic\u2010pituitary axis and mammary gland under HS. Transcriptome analyses by multitissue RNA\u2010Seq indicated the possible mechanisms of reduced lactation performance in animals under HS. In the hypothalamus, the cAMP signaling pathway was activated to resist neuronal death, and the expression of downstream genes was increased to promote cell survival under HS. Reduced food intake might be caused by down\u2010regulated appetite\u2010related peptide, whereas up\u2010regulated neuropeptide Y acted to attenuate reduced food intake. In pituitary, energy stress from lower food intake might result in reduced secretion of prolactin and growth hormone. Under HS, the mammary gland may undergo hypoxic stress, causing mammary epithelial cell apoptosis. Together, these data showed systemic changes in tissues to accommodate the effects of HS on lactation.\u2014Han, J., Shao, J., Chen, Q., Sun, H., Guan, L., Li, Y., Liu, J., Liu, H. Transcriptional changes in the hypothalamus, pituitary, and mammary gland underlying decreased lactation performance in mice under heat stress. 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