Proton magnetic resonance spectroscopy (¹H MRS) provides in vivo biochemical information on tissue metabolites. However, there have been no prior studies using ¹H MRS in dogs with ischemic stroke. The purpose of this study was to investigate the serial metabolic changes of ¹H MRS in the cerebrum of ischemic dogs, and to correlate the metabolic changes with the immunohistochemical features of the ischemic lesion. An ischemic stroke was induced in five health laboratory beagle dogs by permanent middle cerebral artery occlusion using a silicone plug. ¹H MRS was serially performed three times with a 1.5-tesla MR system: before, three days after and 10 days after the stroke. Immunohistochemical staining was performed to determine the expression of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) at both the ipsilateral and contralateral cerebral cortex. Reduced levels of N-acetyl-asparate (P < 0.05), choline (Cho), creatine (Cr) and myo-inositol (mI), and a marked increase in the lactate (Lac) level (P < 0.01) were found at three days after the stroke. At 10 days after the stroke, the increased levels of Lac (P < 0.01) were maintained over time; however, the other metabolites partially recovered. The changes of Cr, Cho and mI were not statistically significant (P > 0.05). There was a significant loss of NeuN and GFAP immunoreactivity at the ischemic core. ¹H MRS may be to a useful diagnostic tool for the evaluation of ischemic stroke in dogs; our results demonstrated that it showed the prominent metabolic features of an ischemic brain.