Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 184.108.40.206) domain, β-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca2+. However, it was not autoprocessed in the presence of Ca2+. Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca2+ suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca2+. We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the Tm value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca2+ and 7.5 °C in the presence of Ca2+, indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C.