Healthy adult male C57BL/6 mice (8 weeks of age) were randomly divided into sham irradiation group (sham) and cranial ir-radiation group (5 Gy  4 d). For the 5 Gy  4 d group, the head of mice were received 20 Gy X-rays (RAD Source RS 2000 series, Suwanee, USA), applied as four doses of 5 Gy in consecutive 4 days at a dose rate of 2.33 Gy/ min. The rest of mouse’s body was completely protected by a 2-cm thick lead shield. For the sham group, the mice were maintained in the same procedure except X-ray irradiation. No radiation leakage through the shield occurred in the present study, as verified by dose measurement of testis under shielding and morphological structure of peripheral main organs. Meanwhile, Monte Carlo simulation was used to calculate the absorbed dose of the peripheral main organs when mice head was irradiated with 20 Gy X-rays. The results showed that the absorbed dose of testis under shielding was 0.018 Gy, which was only 0.9‰ of cranial absorbed dose. Tandem mass tag (TMT) quantitative proteomic analysis of testis at 4 W after cranial irradiation was carried out by LC-BIO Co., Ltd (Hangzhou, China). The differentially expressed proteins (DEPs) were screened and obtained using the cutoffs of fold change > 1.2 and P < 0.05. For bioinformatics analysis, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using the DAVID 6.8 database (https://david.ncifcrf.gov/).