Pro‐Tk‐SP from Thermococcus kodakaraensis consists of the four domains: N‐propeptide, subtilisin (EC 3.4.21.62) 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.