Kisspeptin and neurokinin B (NKB) play a key role in several physiological processes including in puberty, adult reproductive function including the menstrual cycle, as well as mediating the symptoms of menopause. Infundibular kisspeptin neurons, which coexpress NKB, regulate the activity of gonadotropin-releasing hormone (GnRH) neurons and thus the physiological pulsatile secretion of GnRH from the hypothalamus. Outside of their hypothalamic reproductive roles, these peptides are implicated in several physiological functions including sexual behavior and attraction, placental function, and bone health. Over the last two decades, research findings have considerably enhanced our understanding of the physiological regulation of the hypothalamic-pituitary-gonadal (HPG) axis and identified potential therapeutic applications. For example, recognition of the role of kisspeptin as the natural inductor of ovulation has led to research investigating its use as a safer, more physiological trigger of oocyte maturation in in vitro fertilization (IVF) treatment. Moreover, the key role of NKB in the pathophysiology of menopausal hot flashes has led to the development of pharmacological antagonism of this pathway. Indeed, fezolinetant, a neurokinin 3 receptor antagonist, has recently received Food and Drug Administration (FDA) approval for clinical use to treat menopausal vasomotor symptoms. Here, we discuss the roles of kisspeptin and NKB in human physiology, including in the regulation of puberty, menstrual cyclicity, reproductive behavior, pregnancy, menopause, and bone homeostasis. We describe how perturbations of these key physiological processes can result in disease states and consider how kisspeptin and NKB could be exploited diagnostically as well as therapeutically to treat reproductive disorders.