Objective To investigate the relationship between metabolite levels and the time of cerebral ischemia-reperfusion in the left cortex of rats, so as to explore the molecular mechanism of cortical metabolic disorders induced by ischemia-reperfusion injury. Methods Stroke models of focal cerebral ischemia in rats were established by middle cerebral artery occlusion (MCAO). Then a nuclear magnetic resonance (NMR)-based metabolome analytical approach was carried out to analyze the metabolite levels in the left cortex of MCAO rats at different time points (3, 6, and 24 h) after reperfusion. Results Some changes of metabolic pathways, such as energy deficiency, glycolysis aggravation, and neurotransmitter disorders, were observed in the left cortex of MCAO rats at 3 h after reperfusion. All the above-mentioned disorders were alleviated by the autoregulation at 6 h after reperfusion. However, the forementioned metabolic disturbances became severe after 24-hour reperfusion. Conclusion Our results suggest that different extents of metabolic disturbance appears in the cortex at different time points after reperfusion, which might help to understand the molecular mechanism of cerebral ischemia-reperfusion injury, providing reference for regulating metabolic disorders at different time points after stroke in clinic.