In this paper, an NMR-based metabolomic study was applied to unravel the pathological mechanisms of focal cerebral ischemia at the metabolic level by investigating the metabolic profile changes of regional brain tissues of male rats upon MCAO operation. In our study, to induce ischemic defects, the operation of middle cerebral artery occlusion was applied to rats in the model group. Meanwhile, the sham-operation was subjected to the rats in sham group by following the same surgical procedure as that applied to the model group rats without occlusion. Three hours after the operation, the metabolites from regional brain tissues including cortex, hippocampus and striatum from the ischemic left hemisphere and the non-ischemic right hemisphere of experimental rats were extracted and subjected to NMR. Multivariate data analysis of PCA and OPLS-DA methods were then applied to analyze the NMR data and thus unravel the possible correlations between the metabolic profile changes and the variations in biological pathways of MCAO rats. The obtained metabolomic data demonstrated that the neural cell damages and the systematic metabolic disorders including energy deficiency(the decrease in AMP level and the increase in uridine concentration), up-regulation of anaerobic glycolysis(a significant up-regulation of the lactate level), oxidative stress(the up-regulation of either malonate level or succinate concentration), dysfunction of choline metabolism(the significant up-regulation of choline level and the decrease in both GPC level and phosphorylcholine concentration), neurotransmitter imbalances(the down-regulation of glutamate level and the up-regulation of GABA, glycine and alanine concentration), and neuronal cell damage(a decrease in the NAA level), were induced in the regional brain tissues of ischemic left hemispheres of MCAO rats. Moreover, the patterns of the metabolic variations in the non-ischemic hemispheres of MCAO rats were similar to those in the left ones, although the metabolic disorders in the non-ischemic right hemisphere were much less severe. Our results suggest that close attention should be paid to the non-ischemic cerebral regions in the treatment of patients with focal ischemic stroke.