Glyphosate (N-phosphonomethylglycine) is a broad-spectrum, foliar-applied herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants. EPSPS catalyzes a crucial step in the shikimate pathway for the biosynthesis of folates and aromatic amino acids in plants. A variety of glyphosate-tolerant EPSPS enzymes have been reported. Some of these have been introduced into crops using biotechnology to produce glyphosate-tolerant crops. Glyphosate tolerance in crops permits the use of glyphosate to control weeds while maintaining crop yield. We endeavored to optimize the maize EPSPS enzyme with improvements in both enzymatic activity and reduction in sensitivity to glyphosate to improve the potential for herbicide tolerance in crops. Here, we have improved the glyphosate tolerance of maize EPSPS with the potential of providing an herbicide tolerance trait by utilizing enzyme optimization with in vitro and in planta screening. Overexpressing some of these EPSPS variants into maize have resulted in maize plants with robust vegetative glyphosate tolerance.