Microbial food is an important direction of sustainable food development in the future. Microorganisms such as lactic acid bacteria (LAB) are important sources of natural radioprotectors. Moderate environmental stress can induce stress response and improve biological activity of LAB. In the previous study, Lactococcus lactis subsp. lactis IL1403 (L. lactis IL1403) cell-free extract induced by ionizing radiation (IR) of 500 Gy (IR-CFE) presented the stronger radioprotective effect than untreated cell-free extract in mice. To explore the radioprotective active substances of IR-CFE, the key protein was screened by proteomics and its radioprotective effect in vivo was further evaluated. The Kyoto Encyclopedia of Genes and Genomes (KEGG)enrichment showed that two-component systems (TCS) were enhanced to adapt the IR induction. Meanwhile, the protein LlrG belonged to TCS was screened by the protein-protein interaction. Furthermore, the recombinant protein LlrG (rLlrG) could markedly alleviate the 60Coγ-induced damage to the hematopoietic system, oxidative stress and inflammation in mice, thereby exerting its radioprotective function. These results suggested LlrG protein not only played an important role in the adaptation of L. lactis IL1403 to IR environment, but also exerted a good radioprotective effect, which could be applied in the development of protein-based radioprotectors.