Peptidyl-tRNA hydrolase (Pth2) hydrolyzes peptidyl-tRNA, an immature product of aborted translation, into peptide and tRNA, thereby maintaining cellular protein synthesis through peptide release and tRNA recycling. Here, we present the crystal structure of Pth2 from
Candidatus
Lokiarchaeum sp. GC14_75 (
Loki
Pth2) at 2.12 Å resolution. This is the first structure of Pth2 from a lineage within Promethearchaeati, a kingdom of archaea closely related to eukaryotes. The structure reveals that
Loki
Pth2 forms a homodimer and closely resembles Pth2 structures from other species. However,
Loki
Pth2 exhibits two prominent structural differences: a short helix around the catalytic center, which is absent in other Pth2s, and a distinct orientation of the C-terminal helix. Detailed comparative structural analysis suggests that these regions may regulate enzymatic activity and substrate binding, respectively. Furthermore, the corresponding regions in other Pth2s also exhibit high flexibility, suggesting that similar mechanisms may be conserved among Pth2s. To gain insights into the growth environment of
Candidatus
Lokiarchaeum sp. GC14_75, we assess the optimal temperature for the catalytic reaction of
Loki
Pth2, which suggests that
Candidatus
Lokiarchaeum sp. GC14_75 inhabits moderately thermophilic environments.