Hypoxia and ischemia significantly affects perinatal brain development, even worse in preterm infants. However, the details of the mechanism leading to permanent brain damage after hypoxia-ischemia attack have not been fully elucidated. Proteomics could provide insight into the potential mechanism and help to promote the clinical treatment. In this study, quantitative analysis was performed 24 hours after hypoxia-ischemia using liquid-chromatography mass spectrometry coupled to label-free analysis. Compared to control, 193 proteins were present only in hypoxic-ischemic group. In addition, 34 proteins were more than 2 folds up-regulated and 14 proteins were more than 2 folds down-regulated in hypoxia-ischemia group. Gene Ontology database showed that the majority of differentially expressed proteins comprised mitochondrial proteins et al. Molecular function analysis revealed that the majority of proteins were involved in ion binding et al. Biological process analysis showed that the majority of proteins were involved in response to organic substance et al. STRING 9.0 software analysis were used to explore the complex interactions existed among the proteins. Western blot were used to verify the fold changes of some proteins-microtubule-associated protein 2 and microtubule-associated protein tau. This novel study performed a full-scale screening of the proteomics research in hypoxic-ischemic brain damage of immature rat.