Shigella flexneri was the most common pathogen causing bacillary dysentery. Thioredoxin-dependent thiol peroxidase (SF2523) proteins was from Shigella flexneri 2a str. 301. It belonged to thioredoxin peroxidase family and played an important role in protecting the biological macromolecule by scavenging active oxygen generated in the process of aerobic metabolism. To understand the underlying mechanism, prokaryotically expressed thioredoxin-dependent thiol peroxidase protein was purified using affinity chromatography and gel filtration, crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 1.8 mol/L tri-Ammonium citrate, pH 7.0 using 1 g/L protein solution at 289 K. A complete data set was collected from a crystal to 2.75 ? resolution using synchrotron radiation at 100 K. The crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a = 35.80 ?, b = 50.63 ?, c = 88.52 ?, α = β = γ = 90.00°. One molecule was found in the asymmetric unit with a Matthews coefficient of 2.03 ?³/u, corresponding to a solvent content of 39.56%.