β-Galactosidase from Thermus thermophilus A4 (A4-β-Gal) is stable at 70°C for 20 h. It belongs to the glycosyl hydrolase family (GF) 42, and enzymes in GF-42 are stable under extreme circumstances such as high temperature or high salt concentration. In order to obtain the structural basis of its reaction mechanism and stability, we conducted X-ray crystallography of the enzyme. We identified the structure of free and galactose-bound A4-β-Gal at 1.6 A and 2.2 A resolution, respectively. The A4-β-Gal has a “flower pot-like” trimeric structure. The monomer structure of A4-β-Gal is composed of three domains. N-terminal domain (domain A) contains a galactose binding site and has a TIM barrel fold. We identified the residues interacting with the galactose. Since Trp182, a residue related to molecular symmetry, constitutes a part of the active site pocket, the trimeric conformation is necessary for A4-β-Gal to retain enzyme activity. We also identified G1u141 and G1u312 as catalytic residues for A4-β-Gal on the basis of the structural evidence. Glu 141 and G1u312 are located close to the C-termini of the fourth and seventh β-strands of the barrel structure, indicating that GF-42 belongs to 4/7 superfamily.