Over the last two decades, a multitude of gain-of-function studies have been conducted on genes that encode antioxidative enzymes, including one of the key enzymes, manganese superoxide dismutase (). The results of such studies are often contradictory, as they strongly depend on many factors, such as the gene overexpression level. In this study, the effect of altering the ectopic expression level of major transcript variants of the gene on the radioresistance of HEK293T cells was investigated using CRISPRa technology. A significant increase in cell viability in comparison with the transfection control was detected in cells with moderate overexpression after irradiation at 2 Gy, but not at 3 or 5 Gy. A further increase in the level of ectopic expression up to 22.5-fold resulted in increased cell viability detectable only after irradiation at 5 Gy. Furthermore, a 15-20-fold increase in expression raised the clonogenic survival of cells after irradiation at 5 Gy. Simultaneous overexpression of genes encoding SOD2 and Catalase (CAT) enhanced clonogenic cell survival after irradiation more effectively than separate overexpression of both. In conjunction with the literature data on the suppression of the procarcinogenic effects of superoxide dismutase overexpression by ectopic expression of , the data presented here suggest the potential efficacy of simultaneous overexpression of and to reduce oxidative stress occurring in various pathological processes. Moreover, these results illustrate the importance of selecting the degree of overexpression to obtain a protective effect.