Objective To investigate the effect of protein complex, Nogo66 and Freund's adjuvant (FA), on retinal microenvironment in rats with chronic ocular hypertension. Methods After the rat model of chronic high intraocular pressure was established on the right eye (the left eye served as control) with the aid of 532-diode laser, the 18 model rats were equally divided into Nogo66 and ovalbumin groups, receiving vaccination with corresponding agent of 100 μg (in 300 μL FA) on the 1st, 7th and 14th days. The retinal ganglion cells (RGCs) were counted after retrograde labelling with fluorescent gold. The retinal expression of CD3, growth associated protein 43 (GAP43), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were observed by immunofluorescence assay and immunohistochemical staining, and the protein levels of retinal BDNF and GDNF were detected by Western blotting. Results The count of RGCs was significantly lower in the right eyes with chronic ocular hypertension than the left eyes (P < 0.01). Inoculation of Nogo66-FA complex resulted in significantly larger number of RGCs when compared with the rats from the ovalbumin group (P < 0.05). In addition, GAP43 and CD3 were highly expressed in the ganglion cell layer and the outer plexus layer, and BDNF and GNDF in the ganglion cell layer, the inner plexus layer and the inner core layer. The protein levels of BNDF and GDNF in the retina were significantly higher in the Nogo66-FA group than the ovalbumin group (P < 0.05). Conclusion Inoculation of protein complex Nogo66-FA can improve the retinal microenvironment and the activation of T cells, up-regulate the expression of various neurotrophic factors, and increase the count of RGCs in the rat model of chronic ocular hypertension.