In our previous study, we demonstrated that type 2 diabetes affects blood-brain barrier integrity and ultrastructural morphology in Zucker diabetic fatty (ZDF) rats at 40 weeks of age. In the present study, we investigated the possible candidates for diabetes-related proteins in the hippocampus of ZDF rats and their control littermates (Zucker lean control, ZLC), by using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Approximately 2756 protein spots were detected by 2D-DIGE, and an increase or decrease of more than 1.4-fold was observed for 13 proteins in the hippocampal homogenates of ZDF rats relative to those of ZLC rats. Among these proteins, we found four proteins whose levels were significantly lower in the hippocampi of ZDF rats than in those of ZLC rats: glial fibrillary acidic protein (GFAP), apolipoprotein A-I preprotein (apoAI-P), myelin basic protein (MBP), and rCG39881, isoform CRA_a. Among these proteins, apoAI-P protein levels were decreased most prominently in ZDF rats than in ZLC rats, based on Western blot analysis. In addition, immunohistochemical and Western blot studies demonstrated that MBP, not GFAP, immunoreactivity and protein levels were significantly decreased in the hippocampus of ZDF rats compared to ZLC rats. In addition, ultrastructural analysis showed that ZDF rats showed myelin degeneration and disarrangement in the hippocampal tissue. These results suggest that chronic type 2 diabetes affects hippocampal function via reduction of MBP and apoAI-P levels as well as disarrangement of myelin.