In spontaneously ovulating rodent species, the timing of the luteinising hormone (LH) surge is controlled by the master circadian pacemaker in the suprachiasmatic nucleus (SCN). The SCN initiates the LH surge via the coordinated control of two opposing neuropeptidergic systems that lie upstream of the gonadotrophin‐releasing hormone (GnRH) neuronal system: the stimulatory peptide, kisspeptin, and the inhibitory peptide, RFamide‐related peptide‐3 (RFRP‐3; the mammalian orthologue of avian gonadotrophin‐inhibitory hormone [GnIH]). We have previously shown that the GnRH system exhibits time‐dependent sensitivity to kisspeptin stimulation, further contributing to the precise timing of the LH surge. To examine whether this time‐dependent sensitivity of the GnRH system is unique to kisspeptin or a more common mechanism of regulatory control, we explored daily changes in the response of the GnRH system to RFRP‐3 inhibition. Female Syrian hamsters were ovariectomised to eliminate oestradiol (E2)‐negative‐feedback and RFRP‐3 or saline was centrally administered in the morning or late afternoon. LH concentrations and Lhβ mRNA expression did not differ between morning RFRP‐3‐and saline‐treated groups, although they were markedly suppressed by RFRP‐3 administration in the afternoon. However, RFRP‐3 inhibition of circulating LH at the time of the surge does not appear to act via the GnRH system because no differences in medial preoptic area Gnrh or RFRP‐3 receptor Gpr147 mRNA expression were observed. Rather, RFRP‐3 suppressed arcuate nucleus Kiss1 mRNA expression and potentially impacted pituitary gonadotrophs directly. Taken together, these findings reveal time‐dependent responsiveness of the reproductive axis to RFRP‐3 inhibition, possibly via variation in the sensitivity of arcuate nucleus kisspeptin neurones to this neuropeptide.