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The gonadotropin‐releasing hormone—gonadotropin inhibitor (GnRH–GnIH) system in the hypothalamus of mammals is the key factor that controls the entire reproductive system. The aim of this study was to immunolocalize GnIH (RFRP‐3) in the hypothalamus during the estrous cycle and to study the effect of putrescine on the expression of GnRH‐I and GnIH through both in vivo and in vitro (GT1‐7 cells) approach and the circulatory levels of GnRH‐I, GnIH, and gonadotropins were also investigated. The study also aims in analyzing all the immunofluorescence images by measuring the relative pixel count of an image. This study showed the effect of putrescine on the morphology of ovary, uterus, and the expression of the steroidogenic acute regulatory protein in the ovary. This study showed GnIH expression was intense during the diestrus and moderate during proestrus and estrus, whereas mild staining during the metestrus. The study further showed that putrescine supplementation to adult female rats increased both GnRH‐I expression in the hypothalamus as well as the GnRH‐I levels in circulation. The study, for the first time, also showed that putrescine supplementation decreased the expression and release of GnIH. These effects of upregulating GnRH‐I expression and downregulating GnIH expression were confirmed by in vitro experiments using GT1‐7 cells. Putrescine supplementation also increased the gonadotropin levels in the serum. To summarize, putrescine can regulate the hypothalamic–pituitary–gonadal axis by increasing the GnRH‐I, luteinizing hormone, and follicle‐stimulating hormone levels and suppressing GnIH levels. This is the first report showing the simultaneous effects of putrescine on the regulation of both GnRH‐I and GnIH in the hypothalamus.

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