Technical Abstract: We examined the effects of pretreatment with 2-deoxy-D-glucose (2DG) on the middle cerebral artery occlusion/reperfusion (MCAO/R) model in hyperglycemic rats. Proton magnetic resonance imaging and spectroscopy (MRI/MRS) were used to measure the lesion size, the level of cerebral perfusion deficit, and ratio of lactate to N-acetyl aspartate (NAA) in brain regions. By performing sequential diffusion weighted imaging (DWI), gradient echo (GE) bolus tracking, steady state spin echo (SE) imaging and water-suppressed proton MRS techniques, we examined the time course of the early changes of the lactate/NAA peak ratio and perfusion deficit in the hyperglycemic rats undergoing 90 minutes MCAO followed by 24h reperfusion. Compared to the saline treated hyperglycemic rats, 2DG treatment at 10 min prior to MCAO significantly reduced DWI hyper-intensity by ~60% and the lactate/NAA peak ratio by ~70% at 4h after MCAO/R. Both SE measured cerebral blood volume (CBV) and dynamic GE relative cerebral blood flow (CBF) showed that the restoration of blood supply recovered and remained at ~80% of baseline during reperfusion in 2DG treated hyperglycemic rats. These data suggest that inhibition of glucose metabolism by 2DG has a beneficial effect in reducing brain injury and minimizing the production of brain lactate during MCAO/R in hyperglycemic rats.