Objective: Kisspeptin is a neuropeptide that acts mainly on gonadotropin-releasing hormone in the hypothalamus with neurokinin B and dynorphin. Also, its peripheral effects have gained importance in recent years. Klotho is an anti-aging transmembrane glycoprotein with growth hormone and insu lin-like growth factor-1-related effects. Both kisspeptin and klotho seem to interact with prolactin in the hypothalamic-pituitary region. In this study, we aimed to investigate the relationship between hyperprolactinemia and circulating kisspeptin and klotho levels. Methods: In this study, 33 women with hyperprolactinemia and 28 control subjects were included. Blood samples were analyzed for follicle-stimulating hormone, luteinizing hormone, estradiol, pro lactin, α-klotho, kisspeptin, and neurokinin B levels. Results: There was no difference between the groups in terms of kisspeptin and α-klotho levels (P =.68 and P =.11, respectively). Follicle-stimulating hormone and estradiol were significantly lower in the hyperprolactinemia group (P =.02 and P =.03, respectively). Kisspeptin levels were positively correlated with follicle-stimulating hormone levels (r=0.43, P =.01) and α-klotho levels (r = 0.32, P =.01). Neurokinin B was negatively correlated with both kisspeptin and α-klotho values (r = −0.42, P =.01 and r =−0.29, P =.02, respectively). Body mass index was positively correlated with kis speptin and negatively correlated with α-klotho molecule (r = 0.273, P =.03 and r =−0.25, P =.04, respectively). Conclusion: Hyperprolactinemia does not affect circulating kisspeptin and α-klotho levels but posi tively correlates with kisspeptin and α-klotho, which are known to play a role in complex interactions between the hypothalamus and the pituitary that may be the subject of new studies in the future.