Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ₀) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2Å resolution and its complexes with guanine and preQ₀, at 2.3 and 2.5Å resolutions, respectively. The N-terminal catalytic domain folds into an (α/β)₈ barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-{[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl}-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like “PUA domain”, whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an α-helix located at the rim of the (α/β)₈ barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ₀-bound forms. Structural comparison of the ArcTGT·preQ₀, ArcTGT·guanine, and QueTGT·preQ₁ complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.