Objective To study the mechanism of glycerol in treatment of trigeminal neuralgia. Methods After chronic constriction injury was produced to the infraorbital nerve and the model of trigeminal neuralgia in rats was successfully established, the trigeminal ganglion and preganglionic rootlets were exposed through infratemporal fossa approach. In the experimental group, pure glycerol was instilled into the preganglionic rootlets, while in the control group physiologic saline instead. The electrophysiologic response and the results of the electron microscopy were observed at different time points after glycerol injection. Results The latency of sensory evoked potential in the experimental group was longer than that in control group. Under the electron microscope, demyelination and degeneration were very similar in two groups, but disruption of neuraxon was only observed in the experimental group. Neuraxon regeneration occurred 10 weeks later. Conclusion Retrogasserian glycerol rhizotomy in treatment of trigeminal neuralgia was through disruption of myelinated nerve fiber that is reversible.