The serine protease Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1-IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk-subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk-subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen-bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high-temperature environments.