The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, a precursor of the aromatic amino acids and many secondary aromatic metabolites. The shikimate pathway is essential for most bacteria and plants but absent in humans, making it an attractive target for the development of novel antibiotics. 3-phosphoshikimate 1- carboxyvinyltransferase is the sixth enzyme in the shikimate pathway and catalyzes the conversion of shikimate-3-phosphate and phosphoenolpyruvate to 5-enolpyruvyl-3-shikimate phosphate. This protein was co-crystallized with the substrate shikimate-3-phosphate and the commercially successful herbicide glyphosate. The enzyme consists of six βαβαββ motifs, three in both N- and C-terminal domains. The ultrahigh resolution of this structure provides an exquisite view of the mode of shikimate-3-phosphate and glyphosate binding. Glyphosate is observed to bind adjacent to shikimate-3-phosphate in phosphoenolpyruvate’s binding site.