Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H₂DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by ¹³⁷Cs γ radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent.