Ionizing radiation is widely applied in food production as preservation technology and for correction of the gut microbiome of cancer patients, rescuers, astronauts etc. Lactic acid bacteria (LAB) can be used for the same reason. The main goal of this study was to investigate the effect of irradiation on some activities of Lactobacillus rhamnosus MDC 9661 and its effect on the survival of irradiated rats. The results indicate that both ultraviolet (during 45 min) and X‐ray irradiations (with 2 Sv) decreased the CFU and the antibacterial activity of the strain. Higher than 700 Sv dose of X‐ray irradiation resulted in the total inhibition of antibacterial activity with the total reduction of colony forming units less than 10 cells ml−1, while irradiated with 1000 Sv dose L. rhamnosus MDC 9661 did not lose its proteolytic activity. It was also shown that L. rhamnosus MDC 9661 was not immunogenic in the organism of the rats and cannot lead to the development of autoimmune responses. L. rhamnosus MDC 9661 demonstrated the necessary properties for probiotics and can be effectively used for the correction of the gut microbiome of all target groups. The co‐aggregation of the cells is one of the mechanisms for resistance of LAB to irradiation. Significance and Impact of the Study: Ionizing radiation is widely applied for food preservation and for correction of the gut microbiome of patients treated with radiation, rescuers, astronauts, etc. Some lactic acid bacteria (LAB) are of great potential in restoring the microbiome of patients, or in food bio‐preservation. This investigation revealed the radioresistance of Lactobacillus rhamnosus MDC 9661 with only some loss of antibacterial activity, maintaining other activities and the absence of an immunogenic effect. So, it is a potential probiotic organism for the treatment of target people groups. The results showed that co‐aggregation is one of the LAB resistance mechanisms which can be used in future to select effective strains.