The hypothalamic-pituitary-gonadal (HPG) axis governs fertility through the meticulous control of gonadotrophin-releasing hormone (GnRH) pulses. Gonadal sex steroids control the activity of GnRH neurons through feedback regulation, yet the precise cellular and molecular mechanisms governing sex steroid feedback are not fully understood. Kisspeptin is a neuropeptide, a key mediator of sex steroid feedback and a critical stimulus for GnRH secretion. To further elucidate our understanding of kisspeptin in reproduction, we examined the effect of novel kisspeptin antagonists on pulsatile luteinising hormone (LH) secretion. We tested 6 novel and 1 proven kisspeptin antagonists on the LH secretion in ovariectomised (OVX) ewes. We hypothesised that these antagonists will reduce LH secretion, LH pulse amplitude and increase pulse interval during its infusion. OVX ewes were prepared for lateral ventricle (LV) infusion and jugular vein sampling then allocated to either a control group (artificial cerebrospinal fluid, aCSF) or an antagonist (p318, p347, p350, p354, p356, p362, p271) group (n=4 per group). Blood was sampled every 10 min for 3 hours before, 1 hour during and 2.5 hours after an LV infusion of either aCSF (flow rate 200μl/h) or antagonist (300μg/h with an initial 200μg bolus) for plasma LH measurement. Compared to control ewes, the majority of kisspeptin antagonists did not alter mean LH levels or pulse amplitude during the infusion period. The exception was p271, which reduced both mean LH concentration and LH pulse amplitude (both P<0.05). Moreover, antagonists p356, p362, and p271 resulted in significant increases (P<0.05) in LH interpulse interval during the infusion period compared to control. From our study, we can identify potential kisspeptin antagonists in OVX sheep, which offer insights in the role of kisspeptin in this species. These antagonists may also provide unique therapeutic agents for the treatment of hormone-dependent disorders of reproduction.