Background: Glaucoma is the leading cause of irreversible blindness, and the loss of retinal ganglion cells (RGCs) is the most important pathological feature. During the progression of glaucoma, glutamate content in the optic nerve increases, and glutamate-induced excitotoxicity will aggregate the damage and death of RGCs. We have previously reported that olfactory ensheathing cells (OECs) transplantation preserved the visual function of the glaucoma model but the mechanism is unknown. Methods: Adult Long-Evans rats were used in the present study and injecting magnetic microspheres was used to establish a glaucoma model in rats. Optokinetic response test and Pattern electroretinogram recording were used to assess the visual functions of rats. RT-PCR, immunofluorescence, and co-culture experiments were performed to investigate the therapeutic effects and mechanisms of OECs for glaucoma. Results: In the glaucoma model, increased glutamate content and the damage of astrocytes (AC) and RGCs were observed. OECs transplantation reduced the glutamate concentration in the optic nerve, alleviated the apoptosis of AC and RGCs, and protected the visual function of the glaucoma model. Furthermore, we found that OECs possessed a stronger capacity to metabolize excessive glutamate compared with AC and Müller glia. OECs could improve the glutamate microenvironment of the optic nerve to prevent AC and RGCs from glutamate-induced excitotoxicity in glaucoma. And the recovery of AC function further supported the survival of RGCs. Conclusions: We demonstrate that OECs transplantation could play a neuroprotective role by regulating the glutamate microenvironment in glaucoma.