Kisspeptin directly stimulates gonadotropin-releasing hormone (GnRH) release via G protein-coupled receptor 54 (GPR54). This study demonstrates that GPR54 is essential for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in mice and humans. GPR54 transcripts are colocalized with GnRH neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, acts directly on these neurons. GnRH neurons in gpr54-deficient mice appear anatomically normal and project to the median eminence, indicating that hypogonadism in these mice is due to a lack of GnRH release rather than abnormal migration of GnRH neurons. Injection of kisspeptin into gpr54-deficient mice has no effect, showing that kisspeptin acts uniquely via GPR54 signaling for this function. Direct measurement shows that intracerebroventricular administration of kisspeptin in sheep causes a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin occurs directly at the level of GnRH release. These findings define GPR54 as a major control point in the reproductive axis and suggest that kisspeptin is a neurohormonal effector. The study also shows that GPR54 is colocalized with GnRH neurons in the mammalian brain, supporting the hypothesis that GPR54 modulates GnRH release. The results confirm that kisspeptin acts uniquely via GPR54 to stimulate GnRH release, establishing the fundamental role of GPR54 signaling in the physiology of the reproductive axis with implications for reproductive health.Kisspeptin directly stimulates gonadotropin-releasing hormone (GnRH) release via G protein-coupled receptor 54 (GPR54). This study demonstrates that GPR54 is essential for the pubertal onset of pulsatile luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in mice and humans. GPR54 transcripts are colocalized with GnRH neurons in the mouse hypothalamus, suggesting that kisspeptin, the GPR54 ligand, acts directly on these neurons. GnRH neurons in gpr54-deficient mice appear anatomically normal and project to the median eminence, indicating that hypogonadism in these mice is due to a lack of GnRH release rather than abnormal migration of GnRH neurons. Injection of kisspeptin into gpr54-deficient mice has no effect, showing that kisspeptin acts uniquely via GPR54 signaling for this function. Direct measurement shows that intracerebroventricular administration of kisspeptin in sheep causes a dramatic release of GnRH into the cerebrospinal fluid, with a parallel rise in serum LH, demonstrating that a key action of kisspeptin occurs directly at the level of GnRH release. These findings define GPR54 as a major control point in the reproductive axis and suggest that kisspeptin is a neurohormonal effector. The study also shows that GPR54 is colocalized with GnRH neurons in the mammalian brain, supporting the hypothesis that GPR54 modulates GnRH release. The results confirm that kisspeptin acts uniquely via GPR54 to stimulate GnRH release, establishing the fundamental role of GPR54 signaling in the physiology of the reproductive axis with implications for reproductive health.