Glyphosate (Glyph)-based formulations are the most widely used herbicides in the world and their consumption has increased dramatically in recent years. The nervous system results highly vulnerable to a wide spectrum of environmental pollutants that may be linked to the development of brain disorders. In this sense, pesticides exposure has been proposed as the main environmental factor associated with deficits in neurobehavioral performance and neurodegenerative pathologies. Therefore, in the present study we aimed to describe the Glyph effect in the regulation of synaptic assembly in the hippocampus, through in vitro and in vivo assays. Our results reveal that Glyph exposure during a critical period of synaptogenesis decreased dendritic spine density as well as synaptic protein expression, such as PSD-95 and Synapsin-I, in mature cultured neurons. In addition, the exposure of juvenile rats to Glyph reduced PSD-95 protein levels and altered postsynaptic organization in the hippocampus. To associate these abnormalities with cognitive dysfunction we evaluated spatial learning and memory and recognition memory by the Morris water maze and the Novel Object Recognition tests, respectively. We found that Glyph treatment induced memory deficits in both tests compared to controls. Together, these findings suggest that Glyph exposure alters neuronal maturation and synaptic organization impairing normal brain connectivity and complex cognitive behaviour.