Abstract Living organisms are exposed to nitrosative stress mediated by nitric oxide (NO) and its derivatives. Multiple cellular mechanisms may be needed to cope with nitrosative stress. This work takes advantage of a hypersensitive Escherichia coli genetic system to identify genes involved in resistance to nitrosative stress in mouse lungs. Mouse thioredoxin domain-containing 5 (mTrx 5) was identified as one of the candidate genes. Its ability to complement the hypersensitive phenotype in an E. coli mutant strain was confirmed by genetic analysis. Purified recombinant mouse thioredoxin domain-containing 5 protein reduced DNA damage that is sensitive to cleavage by the deamination repair enzyme endonuclease V, indicating that mTrx 5 may play a role in scavenging the reactive nitrogen species. E. coli thioredoxin 1 and thioredoxin 2 proteins also reduced the DNA damage in a similar manner. Deletion of trxA (encodes thioredoxin 1) or trxC (encodes thioredoxin 2) in E. coli resulted in a slightly higher sensitivity to nitrosative stress. On the other hand, deletion of both trxA and trxC greatly increased its sensitivity to nitrosative stress. Complementation with the mTrx 5 gene rescued the sensitive phenotype of the double deletion mutant. The potential roles that mTrx 5 may play in coping with nitrosative stress are discussed.