Widespread application of glyphosate poses a threat to living organisms. Microbial strains are able to degrade glyphosate via contrasting metabolic pathways with the help of enzymes. Glyphosate oxidoreductase (GOX) and C-P lyase are the key enzymes for the biodegradation of glyphosate and its intermediate metabolite aminomethylphosphonic acid (AMPA) in microbes. The microbial degradation of glyphosate has been reported, but the underlying molecular mechanism is still unclear. Therefore, in this study, the interaction mechanism of GOX and C-P lyase with glyphosate and AMPA were investigated by using molecular docking and molecular dynamics (MD) simulations. The results indicate that glyphosate contacts with the active site of GOX and C-P lyase by hydrogen bonds as well as hydrophobic and van der Waals interactions in aqueous solution to maintain its stability. The presence of glyphosate and AMPA in the active site significantly changes the conformation of GOX and C-P lyase. The results of the MD simulations confirm that GOX and C-P lyase complexes are stable during the catalytic reaction. This study offers a molecular level of understanding of the expression and function of GOX and C-P lyase for the bioremediation of glyphosate.