Redox signaling and homeostasis are essential for cell survival and the immune response. Peroxiredoxin (Prx) modulates the level of H2O2 as a redox signal through H2O2 decomposition. The redox activity of thioredoxin (Trx) is required as a reducing equivalent to regenerate Prx. Edwardsiella piscicida is an opportunistic Gram-negative enteric pathogen that secretes a novel Trx-like effector protein, ETAE_2186 (Trxlp). Trxlp has unique structural properties compared with other Trx proteins. In enzymatic and binding assays, we confirmed Trxlp to be redox-inactive due to the low reactivity and flexibility of the resolving cysteine residue, C35, at the active site motif “31WCXXC35”. We identified key residues near the active site that are critical for reactivity and flexibility of C35 by site-directed mutagenesis analysis. NMR titration experiment demonstrated prolong inhibitory interaction of Trxlp with Prx1 resulting in the …