Synapses compute and transmit information to connect neural circuits and are at the basis of brain operations. Alterations in their function contribute to a vast range of neuropsychiatric and neurodegenerative disorders and synapse-based therapeutic intervention, such as selective inhibition of synaptic transmission, may significantly help against serious pathologies. Graphene is a two-dimensional nanomaterial largely exploited in multiple domains of science and technology, including biomedical applications. In hippocampal neurons in culture, small graphene oxide nanosheets (s-GO) selectively depress glutamatergic activity without altering cell viability. Glutamate is the main excitatory neurotransmitter in the central nervous system and growing evidence suggests its involvement in neuropsychiatric disorders. Here we demonstrate that s-GO directly targets the release of pre-synaptic vesicle. We propose that s-GO flakes reduce the availability of transmitter, via promoting its fast release and subsequent depletion, leading to a decline of glutamatergic neurotransmission. We injected s-GO in the hippocampus in vivo, and forty-eight hours after surgery ex vivo patch-clamp recordings from brain slices show a significant reduction in glutamatergic synaptic activity, in respect to saline injections.