Several neurological disorders such as Alzheimer's and Parkinson's diseases have been attributed to gamma-aminobutyric acid (GABA) depletion in the brain. In order to provide a pharmacological basis for the neuroprotective actions of the enhanced accumulation of GABA in mulberry leaves (ML) against cerebral ischemia in vitro and in vivo, a process was developed to enhance the accumulation of GABA in mulberry leaves (GAML) as a result of the various anaerobic treatments. The GABA concentrations were changed by N(2) gas purging, the reaction temperature, reaction time, pH and the leaf size. GABA enhanced the potential of neuroprotection in the PC12 cells damaged by H(2)O(2)-induced oxidation. GAML reduced the cytotoxicity in the PC12 cells against oxygen glucose deprivation-induced cerebral ischemic condition. The neuroprotective effect of GAML was further demonstrated in vivo using middle cerebral artery occlusion brain injury model. GAML significantly decreased the infarct volume of the brain compared with than control group. Overall, these results suggest that the anaerobic treatment of ML makes GAML enhance the neuroprotection effect against in vivo cerebral ischemia such as in vitro.