Diabetes, a risk factor for stroke, leads to elevated blood methylglyoxal (MG) levels. This is due to increased MG generation from the high glucose levels, and because diabetes impairs the glutathione (GSH)-glyoxalase system for MG elimination. MG glycates proteins and causes dicarbonyl stress. We investigated the contribution of MG and GSH to stroke outcome. Cerebral ischemia/reperfusion was performed in chemical-induced (streptozotocin) and genetic Akita mouse models of Type 1 diabetes. Brain infarction and functions of the GSH-dependent MG elimination pathway were determined. Diabetes increased post-ischemia-reperfusion cerebral infarct area in association with elevated MG and diminished GSH levels. Infarct size correlated with brain MG-to-GSH ratio. Expression of glutamate-cysteine ligase catalytic subunit (GCLc) was increased in diabetic brain. GCL activity was unchanged. MG-adducts were elevated in the diabetic brain and, using immunoprecipitation, we identified one of the bands as glycated occludin. This was accompanied by increased blood-brain barrier permeability. Total protein carbonyls were elevated, indicative of oxidative/carbonyl stress. N-acetylcysteine (NAC) corrected MG-to-GSH ratio, and reduced diabetic brain infarct area, occludin glycation and permeability. In addition, protein carbonyls were decreased by NAC. We showed that the diabetic brain exhibited a lower GSH-dependent potential for MG elimination, which contributed to increased protein glycation, and oxidative/carbonyl stress. The consequence of these changes was aggravated post-stroke brain injury. NAC administration protected against the exacerbated brain damage via restored GSH generation and normalization of the MG-to-GSH ratio and possibly by attenuating oxidative/carbonyl stress. This treatment could contribute to the successful management of stroke risk/outcome in diabetes.