Using the electrophysiological method, this paper studied the influences of glyphosate on the nerve impulse generation and conduction of nerve trunk of Bufo gargarizans Cantor. By spraying a glyphosate solution on the toads′ skin, the valid ingredient of glyphosate can penetrate the toads′ bodies and act on their nervous systems. The biological signal recording and processing system was applied to measure the performance parameters of the nerve trunk, including the reaction time, the active potential peak value and the impulse conduction velocity. The results showed that with increasing glyphosate concentration, the reaction time of the nerve trunk got longer, but the peak value of active potential decreased and the velocity of the impulse conduction slowed down. For seven days after being sprayed with glyphosate solution, test toads′ the average reaction time was positively correlated with the spray solution glyphosate concentration. The active potential peak value and impulse conduction velocity were, however, negatively correlated with glyphosate concentration. When Bufo gargarizans were exposed to the ghyphosate solutions in dilutions of 1.64, 2.05, 2.87ml/L, (which are the recommended concentrations for killing field weeds,) the reaction time, active potential peak value and impulse conduction velocity of the tested groups were all statistically different from those of the control group. Meanwhile, the testing time (days after spraying the glyphosate solutions) also affected the action potential generation and conduction of the nerve trunk. The reaction time of the nerve trunk was positively correlated with testing time. However, the relationship between the impulse conduction velocity and the testing time was negative. It can be concluded that the application of glyphosate will decrease the sensitivity of the nerve trunk and also hamper the impulse generation and conduction of Bufo gargarizans.