The CutA1 protein from Pyrococcus horikoshii (PhCutA1), a hyperthermophile, has an unusually high content of charged residues and an unusually high denaturation temperature. To elucidate the role of ion–ion interactions in protein stability, mutant proteins of PhCutA1 in which charged residues were substituted by noncharged residues were comprehensively examined. The denaturation temperatures (Td) for 13 of 53 examined mutant proteins were higher than that of the wild-type (148.5 °C at pH 7.0), among which E99Q had the highest Td at 154.9 °C. R25A had the largest decrease in Td among single mutants at ΔTd = −12.4 °C. The average decrease in Td of Lys or Arg mutants was greater than that of Glu or Asp mutants, and the average change in Td (ΔTd) of 21 Glu mutants was negligible, at 0.03 ± 2.05 °C. However, the electrostatic energy (−159.3 kJ·mol−1) of PhCutA1 was quite high, compared with that of CutA1 from Escherichia coli (−9.7 kJ·mol−1), a mesophile. These results indicate that: (a) many Glu and Asp residues of PhCutA1 should be essential for highly efficient interactions with positively charged residues and for generating high electrostatic energy, although they were forced to be partially repulsive to each other; (b) the changes in stability of mutant proteins with a Td value of ∼ 140–150 °C were able to be explained by considering factors important for protein stability and the structural features of mutant sites; and (c) these findings are useful for the design of proteins that are stable at temperatures > 100 °C.