Chronic stress can induce hippocampus injury such as neuron loss, dendrite atrophy, but its mechanism and molecular basis remain unclear up to now. To understand the molecular mechanism on protein level and find the crucial proteins which correlated with chronic stress-induced injury, two-dimensional electrophoresis was applied to separate the hippocampal total proteins of control group and restraint stressed rats, then the differential expressed proteins were detected by image analysis and identified by matrix assisted laser desorption /ionization time of flight mass spectrometry (MALDI-TOF-MS) as well as database searching. Moreover, the 2-DE results were verified on the mRNA level by semi-quantitative RT-PCR. The hippocampal 2-DE map with high resolution and good reproducibility of control and stress group rats were obtained. Fourteen differentially expressed protein spots were detected and eleven proteins were successfully identified, most of these proteins were involved in the process of energy metabolism and signal transduction. These results provide a clue for elucidating the mechanism of chronic stress-induced hippocampal injury and are useful for elevating the adaptability to stress.