Hippocampal sclerosis (HS) is the most common neuropathological condition in adults with drug-resistant epilepsy and represents a critical feature in mesial temporal lobe epilepsy (MTLE) syndrome. Although epileptogenic brain tissue is associated with glutamate excitotoxicity leading to oxidative stress, the proteins that are targets of oxidative damage remain to be determined. In the present study we designed comprehensive analyses of changes in protein expression level and protein oxidation status in the hippocampus or neocortex to highlight proteins associated with excitotoxicity by comparing MTLE patients with relatively mild excitotoxicity (MTLE patients without HS, MTLE-non-HS) and those with severe excitotoxicity (MTLE patients with HS, MTLE-HS). We performed 2-dimensional fluorescence difference gel electrophoresis, 2D-oxyblot analysis, and mass spectrometric amino acid sequencing. We identified 16 proteins at 18 spots in which the protein expression levels differed between sclerotic and non-sclerotic hippocampi. In the sclerotic hippocampus, the expression levels of several synaptic proteins were decreased, and those of some glia-associated proteins increased. We confirmed histologically that all MTLE-HS cases examined exhibited severe neuronal cell loss and remarkable astrocytic gliosis in the hippocampi. In all MTLE-non-HS cases examined, neurons were spared and gliosis was unremarkable. Therefore, we consider that decreased synaptic proteins are a manifestation of loss of neuronal cell bodies and dendrites, whereas increased glia-associated proteins are a manifestation of proliferation and hypertrophy of astrocytes. These are considered to be the result of hippocampal sclerosis. In contrast, the expression level of d-3-phosphoglycerate dehydrogenase (PHGDH), an l-serine synthetic enzyme expressed exclusively by astrocytes, was decreased, and that of stathmin 1, a neurite extension-related protein expressed by neurons, was increased in the sclerotic hippocampus. These findings cannot be explained solely as the result of hippocampal sclerosis. Rather, these changes can be involved in the continuation of seizure disorders in MTLE-HS. In addition, the protein carbonylation detection, an indicator of protein oxidation caused by excitotoxicity of multiple seizures and/or status epilepticus, revealed that the carbonyl level of collapsin response mediator protein 2 (CRMP2) increased significantly in the sclerotic hippocampus. In conclusion, protein identification following profiling of protein expression levels and detection of oxidative proteins indicated potential pathognomonic protein changes. The decreased expression of PHGDH, increased expression of stathmin 1, and carbonylation of CRMP2 differentiate between MTLE with and without HS. Therefore, further investigations of PHGDH, stathmin 1 and CRMP2 are promising to study more detailed effects of excitotoxicity on epileptogenic hippocampal tissue.