The widespread use of herbicides, including glyphosate (Gly), is thought to represent a potentially underestimated risk to human health. The putative absence of the known direct target molecular pathway of Gly in mammalian cells has assured its safety and EFSA established the acceptable daily intake (ADI) dose for humans at 0,5 mg/kg body weight. However, very little is known about Gly neurotoxicity at this dosage, especially on intricate mechanism such as the communication between brain cells. Astrocytes contribute to neuron maturation, survival, and modulation of synaptic activity also through the release of extracellular vesicles (EVs). Due to their positioning at the blood-brain barrier (BBB), astrocytes are likely directly affected by Gly crossing the BBB. In a parallel study, we demonstrate that exposing neurons to ADI of Gly produces severe alterations in inhibitory synaptic transmission. Here, we explored whether Gly can alter astrocytes-neurons communication and if this mechanism is mediated by EVs. We found that ADI/Gly produces both an increase of reactive astrocytes in-vitro and a higher release of EVs (Gly-EVs) compared to their control group. Furthermore, cortical neurons exposed to Gly-EVs showed a robust reduction of Homer1bc+ dendritic spines. The electrophysiological analysis of neural network activity and synaptic transmission will define the functional impact of astrocytes-neurons communication mediated by Gly-EVs. Overall, we hope that this work will contribute to better clarify if the use of Gly poses real risks to animal and human health that are yet to be identified.