Objective：To investigate the differentially expressed proteins between mouse testicular tissue with ionizing radiation injury and normal testicular tissue using bioinformatics methods and the functions and biological processes of these proteins. Methods：A mouse model of ionizing radiation injury was established. Comparative proteomic methods，isobaric tags for relative and absolute quantitation，and liquid chromatography-tandem mass spectrometry were used to screen out differentially expressed proteins，and then David and Uniport databases were used for gene ontology（GO） and Kyoto Encyclopedia of Genes and Genome（KEGG） enrichment analyses of differentially expressed proteins. Results：A total of 1633 proteins were identified in mouse testicular tissue with ionizing radiation injury and normal testicular tissue，with 128 differentially expressed proteins in total，among which 28 were upregulated and 100 were downregulated. GO analysis showed that the differentially expressed proteins were mainly involved in biological processes such as oxidation-reduction process，spermatogenesis，and ATP metabolism. KEGG analysis showed that these proteins were mainly distributed in 21 signaling pathways including Parkinson’s disease，oxidative phosphorylation，and metabolic pathways，etc. Conclusion：The bioinformatics method is used for data mining of proteins in mouse testicular tissue with ionizing radiation injury and normal testicular tissue，which provides a reference for further research on the mechanism of ionizing radiation in inducing testicular tissue injury and new biomarkers.