Homoisocitrate dehydrogenase (HICDH) is involved in the α-aminoadipate pathway of lysine biosynthesis in some bacteria and higher fungi, and catalyses the oxidative decarboxylation of (2R,3S)-homoisocitrate into 2-ketoadipate using NAD+ as a coenzyme. In this study, the crystal structure of Thermus thermophilus HICDH in a binary complex with a designed inhibitor, (2S,3S)-thiahomoisocitrate, has been determined at 2.6 Å resolution. The inhibitor observed as a decarboxylated product interacts through hydrogen bonding to Arg 118, Tyr 125 and Lys 171 in the active site. The induced fitting was also observed around the region consisting of residues 120–141, which shifted up to 2.8 Å towards the active site. In addition, it was found that the complex structure adopts a closed conformation in two domains. While the structure of apo-HICDH shows that a catalytic residue Tyr 125 and Arg 85 that engages in substrate recognition are flipped out of the active site, these residues turn towards the active site in the complex structure. The results revealed that they directly interact with a substrate and are involved in catalysis or substrate recognition. Furthermore, by comparing the binary complex with the quaternary complex of Escherichia coli isocitrate dehydrogenase, the substrate recognition mechanism of HICDH is also discussed.