Bacterial resistance to β-lactams is mainly due to the production of β-lactamase. Especially through the production of extended-spectrum β-lactamases (ESBLs), bacteria have acquired resistance not only to penicillins, but also to expanded-spectrum cephems. Here, we describe the crystal structure of the E166A mutant of class A β-lactamase Toho-1 at 1.8 Å resolution, the first reported tertiary structure of an ESBL. Instead of the wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alanine, was used for this study, because of the strong tendency of the wild-type enzyme to form twinned crystals. The overall structure of Toho-1 is similar to the crystal structures of non-ESBLs, with no pronounced backbone rearrangement of the framework. However, there are some notable local changes. First, a difference in the disposition of an arginine residue, which is at position 244 in non-ESBLs but at position 276 in Toho-1 and other ESBLs, was revealed and the role of this arginine residue is discussed. Moreover, changes in the hydrogen-bonding pattern and in the formation of the hydrophobic core were also observed near the Ω loop. In particular, the lack of hydrogen bonds in the vicinity of the Ω loop could be a cause of the extended substrate specificity of Toho-1. Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurring on complex formation is discussed based on the active-site cleft structure and the substrate profile.