Glaucoma is a neurodegenerative disease of the visual system. While elevated intraocular pressure is considered to be a major risk factor, the primary cause and pathogenesis of this disease are still unclear. This study aims to employ in vivo proton magnetic resonance spectroscopy ((1)H MRS) to evaluate the metabolic changes in the visual cortex in a rat model of chronic glaucoma. Five Sprague-Dawley female rats were prepared to induce ocular hypertension unilaterally in the right eye by photocoagulating the episcleral and limbal veins using an argon laser. Single voxel (1)H MRS was performed on each side of the visual cortex 6 weeks after laser treatment. Relative to the creatine level, the choline level was found to be significantly lower in the left glaucomatous visual cortex than the right control visual cortex in all animals. In addition, a marginally significant increase in glutamate level was observed in the glaucomatous visual cortex. No apparent difference was observed between contralateral sides of the visual cortex in T1-weighted or T2-weighted imaging. The results of this study showed that glaucoma is accompanied with alterations in the metabolism of choline-containing compounds in the visual cortex contralateral to the glaucomatous rat eye. These potentially associated the pathophysiological mechanisms of glaucoma with the dysfunction of the cholinergic system in the visual pathway. (1)H MRS is a potential tool for studying the metabolic changes in glaucoma in vivo in normally appearing brain structures, and may possess direct clinical applications for humans. Measurement of the Cho:Cr reduction in the visual cortex may be a noninvasive biomarker for this disease.