Glyphosate is the most widely used herbicide in the world which is sometimes utilized as a pre-harvest desiccant during wheat cultivation. It inhibits the 5-enolpyruvylshikimate-3-phosphate synthase enzyme in the shikimic acid pathway in plants. Although this pathway is not found in humans, it is present in human gut microbiota. In this context, the goal of this study was to examine the effect of pre-harvest glyphosate application on wheat quality, biochemical characteristics and human gut microbiota. The results of this study indicated that the effects of glyphosate on wheat quality is more pronounced when applied at soft dough stage. Glyphosate lowered B-type starch granules and vice versa for A-type granules and it changed the proportions of rapidly digestible and slowly digestible starch. Starch amylopectin chain length distribution was also impacted although the effects were different when applied at the two stages. Glyphosate lowered the molecular weight of SDS extractable and unextractable proteins when applied as a desiccant. Additionally, shikimic acid accumulation was especially high in samples treated at soft dough stage. As for gut microbiota, the results indicated that glyphosate may not have a profound impact on metabolite production by gut microbiota, although there maybe effects on bacterial population dynamics. Overall, the current study indicates that glyphosate applied pre-harvest has some effects on wheat physicochemical properties and gut microbiota. In the context of wheat chemistry, the effects of glyphosate on the shikimic acid pathway, followed by subsequent accumulation of shikimic acid and effects on carbon flow may cause changes in the biosynthesis of starch and proteins. Glyphosate could impact enzyme activity, as it can interact with metals that are required as co-factors in enzyme catalyzed reactions. Glyphosate’s effect on intermolecular interactions between starch and protein, and other macromolecules such as dietary fiber, may also influence the overall chemistry of plant components. Although the effects of glyphosate on gut microbiota are not clear-cut, this exploratory study is a stepping stone in this area of research. In conclusion, the observations made in this study should be investigated further to determine causal links and relationships.