The acclimation process of activated sludge from a wastewater treatment plant for degradation of glyphosate and its biodegradation kinetics were studied in a batch reactor. The parameters monitored included the concentrations of glyphosate, as well as aminomethylphosphonic acid (AMPA), its main metabolite, total organic carbon (TOC), pH, dissolved oxygen (DO) and biomass concentration. At the end of the acclimation process, glyphosate removal efficiency of the acclimated sludge was compared to the fresh sludge one. The results showed that the acclimation process highly increased degradation efficiency. Complete glyphosate removal has been achieved during kinetics experiments. Glyphosate removal kinetic of the acclimated sludge was modeled by Monod model that accurately fitted the experimental results with a maximum growth rate (μmax) of 0.34 h−1 and half-saturation constant (Ks) of 1600 mg L−1. Finally, a biodegradation pathway of glyphosate used as carbon source was proposed.