Glyphosate (Gly)-based herbicides (GBH), which are widely used worldwide, work by inhibiting the enzyme enolpyruvylshikimate-3-phosphate synthase, in the shikimate pathway, which functions in plants but not animals. Long-term exposure to GBH can cause toxic effects to the nervous system by affecting neurotransmitter expression, oxidative stress, anxiety, learning and memory impairments. Though Gly safety studies are still ongoing, little is known about the mechanistic processes underlying Gly neurotoxicity. Remarkably, few studies examined how the acceptable daily intake (ADI) dosage affects neurons. To bridge this gap, we investigated the effects of Gly administration, at ADI concentration, on both function and structure of synapses in primary hippocampus neuronal cells. Both confocal imaging of immunolabelled neurons and patch-clamp recordings disclosed that 30’ Gly administration (3mM) affects the integrity of GABAergic, but not glutamatergic, neurotransmission. Both frequency and amplitude of miniature inhibitory postsynaptic currents decreased, suggesting both pre- and post-synaptic effects of Gly. Moreover, our data showed that Gly reduces the number of release sites, the size of the ready-releasable pool of synaptic vesicles, and the number of postsynaptic ionotropic GABAA receptors. In Gly-treated neurons, morphological investigations revealed a decreased density of both pre-synaptic (vGAT+) and post-synaptic (gephyrin+) inhibitory compartments, while excitatory contacts showed no major changes. In conclusion, our data disclose previously unidentified neuronal and synaptic abnormalities resulting from exposure to the ADI dose of Gly. These findings warn that further in-depth exploration on Gly neurotoxicity is an urgent public-health issue, in order to reveal the intricate molecular and cellular pathways underlying these effects.