Glyphosate-based herbicides (GBH) are the most used herbicides in the world. The human gut microbiome may be exposed to glyphosate via residues in human nutrition. The herbicide inhibits the production of vital aromatic amino acids by targeting the EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) enzyme in the Shikimate pathway, almost universally present in plants, fungi, and bacteria. EPSPS enzymes occur in glyphosate sensitive or resistant forms. In bacteria, these differences in the glyphosate sensitivity may lead to a dysbiosis of the gut microbiome after a long-term exposure to the herbicide, which could have pervasive effects on human health. I assessed the glyphosate susceptibility of common human gut bacteria by analyzing 890 EPSPS protein sequences belonging to 101 species, by using a new bioinformatic tool. Moreover, I cultured five bacterial strains (Escherichia coli IHE3034, Salmonella enterica SL1344, Shigella flexneri M90T, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) and treated them with pure glyphosate and a glyphosate manufacture Roundup Flex. The results indicate that 54% of the common gut bacteria species only had glyphosate sensitive strains (including genera such as Escherichia and Faecalibacterium) and 29% only had resistant strains (including genera Helicobacter and Dorea). Moreover, 7% of the species showed intraspecific variation, and 10% had EPSPS sequences that are yet to be classified. Cell cultures showed that bacteria growth depends on species and glyphosate manufacture. The pathogenic bacteria S. enterica SL1344 showed high tolerance to both glyphosate manufactures. In conclusion, the intrinsic sensitivity to glyphosate is variable among gut bacteria. Moreover, additional indirect mechanisms participate in the sensitivity to glyphosate and should be considered in future studies. Thus, the presence of glyphosate in human food may accentuate this variation, leading to 1) a dominance of tolerant and easily adapting bacteria, and 2) a decrease of diversity in the human gut microbiome. Further studies are needed to investigate the amount of glyphosate in human gut and its effects on microbial communities, as well as to human health.