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Glyphosate-based herbicides are the most used herbicides in the world. Despite being widely used, a dispute exists whether glyphosate-based herbicides have a negative effect on human health, particularly genotoxic effects. Therefore, the aim of this study was to investigate glyphosate genotoxicity on cultured human lymphocytes. Cultured human lymphocytes were treated with different concentrations of glyphosate (20, 40, and 200 µmol/L). Four toxicity measures were examined: frequency of chromosomal aberrations (CAs), frequency of sister-chromatid exchange (SCE), production of 8-OHdG, and cell kinetics analysis. The results show that glyphosate induced significant ( P < 0.05) increases in the levels of SCE at the highest used concentration (200 μmol/L). However, no significant elevation in SCE levels was observed at the lower examined concentrations (20 and 40 μmol/L). No significant changes in CA were detected at all examined concentrations ( P = 0.86). Also, glyphosate did not induce changes to the normal level of 8-OHdG at all examined concentrations ( P = 0.98). Last, no significant changes in either mitotic index or proliferative index were observed at any examined concentrations ( P > 0.05). The results collectively indicate a lack of genotoxicity and cytotoxicity of glyphosate in cultured human lymphocytes when dealing with environmentally relevant concentrations (20 and 40 μmol/L). However, being exposed to higher concentrations (200 μmol/L) led to slightly higher level of SCE. Therefore, we recommend cautionary measures when dealing with glyphosate-based herbicides for individuals, such as farmers, who may be extensively exposed to high concentrations of these herbicides.