Objective   To screen differentially expressed proteins (DEPs) in the hippocampal tissues of rats with chronic diffuse axonal injury (DAI), in order to provide an important theoretical basis for exploring the potential pathogenesis of chronic DAI as well as for clinical diagnosis, screening of drug therapeutic targets, and assessment of prognosis.  Methods   The experimental animals were divided into model group (DAI group, n=20) and control group (CON group, n=20). A modified Marmarou method  was used to establish a DAI model in SD rats. After three weeks of modeling, the changes in protein profiles of hippocampal tissues between two groups were compared with the 4D-Label-free technology, and DEPs were screened by the fold change(FC) in expression of the DAI group/CON group of >1.2 or <0.83 with P<0.05. Bioinformatical analysis of selected DEPs was performed by using gene ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genes (KEGG) pathway enrichment analysis method.  Results   A total of 92 DEPs were screened with 52 upregulated and 40 downregulated. The result  of GO analysis showed that DEPs were mainly involved in biological process (BP) functions such as dephosphorylation, ATP synthesiscoupled electron transfer, positive regulation of hydrogen peroxide-mediated programmed cell death, and internalization of neurotransmitter receptors. The results of KEGG pathway analysis suggested that DEPs were mainly involved in signaling pathways such as metabolic pathways, reactive oxygen species, neurodegenerative pathwaysmultiple diseases, intraretrograde cannabinoid signaling, and glutathione metabolism.  Conclusion   The DEPs in hippocampus of chronic DAI group rats are screened by 4D-Label-free technique. The selected DEPs and their enriched BP and signaling might pathway provide theoretical basis for further study of chronic DAI.