The novel hypothalamic peptides avian gonadotropin inhibitory hormone (GnIH) and its mammalian analogue RFRP-3, are emerging as key negative regulators of reproductive functions across species. GnIH/RFRP-3 reduces gonadotropin release and may play an inhibitory role in ovulation and seasonal reproduction, actions opposite to that of the puberty-promoting kisspeptins. GnIH directly inhibits gonadotropin release from the anterior pituitary in birds. GnIH/RFRP-3-immunoreactive fibres also abut the preoptic-septal gonadotropin-releasing hormone (GnRH) neurons, suggesting an additional site of action that has not been studied at the cellular level. Using anatomical labelling and electrophysiological recordings in septal brain slices from GnRH-GFP, vGluT2-GFP and GAD67-GFP mice, we report inhibitory actions of GnIH/RFRP-3 on kisspeptin-activated vGluT2 (vesicular glutamate transporter 2)-GnRH neurons as well as on kisspeptin-insensitive GnRH neurons, but not on cholinergic or GABAergic neurons (n = 531). GnIH and RFRP-3 produced a strikingly similar non-desensitizing hyperpolarization following brief 15 s applications (GnIH: 9.3 +/- 1.9 mV; RFRP-3: 9.0 +/- 0.9 mV) with IC(50) values of 34 and 37 nm, respectively. The inhibitory effect was mediated via a direct postsynaptic Ba(2+)-sensitive K(+) current mechanism and could prevent or interrupt kisspeptin-induced activation of vGluT2-GnRH neurons. GnIH-immunoreactive fibres were in apparent contact with vGluT2-GFP neurons. Thus, GnIH/RFRP-3 could reduce GnRH and glutamate release in target brain regions and in the median eminence via a direct inhibition of vGluT2-GnRH neurons. This in turn could suppress gonadotropin release, influence reproductive development and alter sex behaviour.