In the mammalian central nervous system (CNS) GABA A receptors (GABA A Rs) mediate neuronal inhibition and are important therapeutic targets. GABA A Rs are composed of 5 subunits, drawn from 19 proteins, underpinning expression of 20–30 GABA A R subtypes. In the CNS these isoforms are heterogeneously expressed and exhibit distinct physiological and pharmacological properties. We report the discovery of S44819, a novel tricyclic oxazolo-2,3-benzodiazepine-derivative, that selectively inhibits α5-subunit-containing GABA A Rs (α5-GABA A Rs). Current α5-GABA A R inhibitors bind to the “benzodiazepine site”. However, in HEK293 cells expressing recombinant α5-GABA A Rs, S44819 had no effect on 3H-flumazenil binding, but displaced the GABA A R agonist 3H-muscimol and competitively inhibited the GABA-induced responses. Importantly, we reveal that the α5-subunit selectivity is uniquely governed by amino acid residues within the α-subunit F-loop, a region associated with GABA binding. In mouse hippocampal CA1 neurons, S44819 enhanced long-term potentiation (LTP), blocked a tonic current mediated by extrasynaptic α5-GABA A Rs, but had no effect on synaptic GABA A Rs. In mouse thalamic neurons, S44819 had no effect on the tonic current mediated by δ-GABA A Rs, or on synaptic (α1β2γ2) GABA A Rs. In rats, S44819 enhanced object recognition memory and reversed scopolamine-induced impairment of working memory in the eight-arm radial maze. In conclusion, S44819 is a first in class compound that uniquely acts as a potent, competitive, selective antagonist of recombinant and native α5-GABA A Rs. Consequently, S44819 enhances hippocampal synaptic plasticity and exhibits pro-cognitive efficacy. Given this profile, S44819 may improve cognitive function in neurodegenerative disorders and facilitate post-stroke recovery.