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Glyphosate is widely used worldwide as a potent herbicide. Due to its ubiquitous use, it is detectable in air, water and foodstuffs and can accumulate in human biological fluids and tissues representing a severe human health risk. In plants, glyphosate acts as an inhibitor of the shikimate pathway, which is absent in vertebrates. Due to this, international scientific authorities have long-considered glyphosate as a compound that has no or weak toxicity in humans. However, increasing evidence has highlighted the toxicity of glyphosate and its formulations in animals and human cells and tissues. Thus, despite the extension of the authorization of the use of glyphosate in Europe until 2022, several countries have begun to take precautionary measures to reduce its diffusion. Glyphosate has been detected in urine, blood and maternal milk and has been found to induce the generation of reactive oxygen species (ROS) and several cytotoxic and genotoxic effects in vitro and in animal models directly or indirectly through its metabolite, aminomethylphosphonic acid (AMPA). This review aims to summarize the more relevant findings on the biological effects and underlying molecular mechanisms of glyphosate, with a particular focus on glyphosate's potential to induce inflammation, DNA damage and alterations in gene expression profiles as well as adverse effects on reproduction and development.