Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P3(1) or P3(2), with unit-cell parameters a = b = 92.1, c = 232.6 Å.