The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery-derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurons is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO and appropriate bicarbonate-free artificial CSF, we investigated the role of the neuron-specific K⁺/Cl^- cotransporter, KCC2, in regulating efficacy of the GABA_A receptor-mediated inhibitory response at the SFO-MnPO synapse. When somata of the MnPO neurons was loaded with 18 mM chloride, stimulation of the SFO evoked outward IPSCs in 81% of the MnPO neurons held at -60 mV. Accordingly, E_IPSC was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl^- extrusion system in these neurons. E_IPSC estimated with gramicidin-based perforated-patch recordings amounted -89.2 ± 4.3 mV. Furosemide (100µM), a pharmacological compound known to block the activity of the neuron-specific K⁺/Cl^- cotransporter, KCC2, reversed IPSC polarity and shifted E_IPSC towards theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2 positive intermingled fibers. In the presence of a GABA_B receptor antagonist, high frequency stimulation (5Hz) of the SFO evoked a train of IPSCs or IPSPs, whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5Hz stimulation carried out in the presence of furosemide (50µM) evoked IPSCs/IPSPs, whose amplitude collapsed during the high-frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurons lacking the functional Cl^- extrusion mechanism. We conclude that a majority of MnPO neurons were characterized by a functional Cl^- transporter that insured efficient activity-dependent Cl^- transport rate, allowing sustained synaptic inhibition of these neurons. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmission afferent to the MnPO, a key-structure in the physiology of the hydromineral and cardiovascular homeostasis.