Gamma irradiation causes severe cellular damage through indirect oxidative stress generated by water radiolysis. Although carotenoids are well known as antioxidant pigments, their independent contribution to microbial resistance against highly penetrating ionizing radiation remains to be investigated. In this study, recombinant Escherichia coli was used as a controlled and radiation-sensitive host to evaluate whether engineered carotenoid accumulation improves gamma-radiation tolerance and protects DNA from irradiation-induced structural damage. Lycopene- and β-carotene-producing E. coli strains were compared with non-carotenoid-producing controls, including an empty-vector control grown under the same carotenoid-inducing conditions. Under carotenoid-producing conditions, the D₁₀ values of the lycopene- and β-carotene-producing strains significantly increased compared with the control strain. To examine the chemical contribution of carotenoids to DNA protection, purified pUC19 plasmid DNA was irradiated in the presence or absence of carotenoids. Agarose gel electrophoresis showed that lycopene and β-carotene reduced the conversion of supercoiled DNA to open-circular and linear forms, while AFM imaging confirmed improved preservation of intact plasmid morphology. These findings suggest that carotenoid biosynthesis may be applied as a metabolic engineering strategy to improve microbial robustness in irradiation-associated biotechnology, food microbiology, environmental applications, and stress-tolerant production platforms.